DNA polymerase with increased gene mutation specificity

ABSTRACT

A DNA polymerase in which a mutation is induced at a specific amino acid position to increase gene mutation specificity, a nucleic acid sequence encoding the polymerase, a vector comprising the nucleic acid sequence, and a host cell transformed with the vector are disclosed. Provided are a method for in vitro detecting one or more gene mutations or SNPs in one or more templates by using a DNA polymerase having increased gene mutation specificity, a composition for detecting a gene mutation or SNP comprising the DNA polymerase, and a PCR kit comprising said composition. Furthermore, provided are a PCR buffer composition for increasing the activity of a DNA polymerase having increased gene mutation specificity and a PCR kit for detecting a gene mutation or SNP comprising the PCR buffer composition and/or the DNA polymerase having increased gene mutation specificity.

TECHNICAL FIELD

The present invention relates to a DNA polymerase with increased genevariation specificity and a PCR buffer composition for increasing theactivity thereof, and more specificity, a DNA polymerase with increasedgene variation specificity due to a mutation occurring at a specificamino acid position, a nucleic acid sequence encoding the polymerase, avector including the nucleic acid sequence and a host cell transformedwith the vector, a method of in vitro detecting one or more genevariations or SNPs in one or more templates using the DNA polymerasewith increased gene variation specificity, a composition for detecting agene variation or SNP, which includes the DNA polymerase, and apolymerase chain reaction (PCR) kit including the composition.

Moreover, the present invention provides a PCR buffer composition forincreasing the activity of the DNA polymerase with increased genevariation specificity, a PCR kit for detecting a gene variation or SNP,which includes the PCR buffer composition and/or the DNA polymerase withincreased gene variation specificity, and a method of in vitro detectingone or more gene variations or SNPs in one or more templates using thekit.

BACKGROUND ART

Since the first human genomic sequence has been defined, the inventorshave focused on finding the genetic difference among individuals, suchas single nucleotide polymorphisms (SNPs). SNPs in a genome are ofinterest because it is more and more clear that they are associated withdifferent drug resistances or predisposing factors for various diseases.Due to the subsequent knowledge of medically-related nucleotidevariation, a therapeutic method for the genetic supply of an individualmay be applied, and a drug therapy which is ineffective or causes a sideeffect may be prevented. The development of technology that enablestime- and cost-effective identification of nucleotide variation willbring further advances in pharmacogenetics.

SNPs account for the major genetic variations in a human genome andcause 90% or more of differences between individuals. To detect othernucleic acid variations such as the genetic variations and mutations,various methods may be used. For example, the identification of avariant of a target nucleic acid may be accomplished by hybridizing anucleic acid sample to be analyzed with a hybridization primer specificfor a sequence variant under suitable hybridization conditions.

However, it was found that such a hybridization method cannot satisfyclinical needs, particularly, in terms of sensitivity, which is requiredfor an assay. Therefore, PCR has been extensively used in molecularbiology and a diagnostic testing method for detecting mutations such asSNPs and other allelic sequence variants. Here, in consideration of thepresence of a variant, a target nucleic acid to be tested was amplifiedby polymerase chain reaction (PCR) before hybridization. As ahybridization probe for the assay, generally, a single-strandedoligonucleotide is generally used. A modified embodiment of the assayincludes a fluorescent hybridization probe. Generally, efforts have beenmade to automate methods of measuring SNPs and other sequence variations(Gut, Hum. Mutat. 17, 475-492 (2001)).

An alternative to sequence variation-specific hybridization known in theart is provided by so-called gene variation-specific amplification. Inthis detection method, during amplification, a variation-specificamplification primer is used, and generally has a so-called differentialterminal nucleotide residue at the 3′ end of the primer, where theresidue is only complementary for one specific variation of a targetnucleic acid to be detected. In this method, the nucleotide variant ismeasured by the presence or absence of a DNA product after PCRamplification. The principle of gene variation-specific amplification isbased on the formation of a canonical or non-canonical primer-templatecomplex at the end of a gene variation-specific amplification primer.Precisely, at the 3′ end of the paired primer, amplification occurs by aDNA polymerase, but at the mismatched primer end, extension issuppressed.

For example, U.S. Pat. No. 5,595,890 discloses a method for genevariation-specific amplification and its application thereof, forexample, the application to detect clinically associated point mutationin a k-ras tumor gene. In addition, U.S. Pat. No. 5,521,301 discloses anallele-specific amplification method for genotyping of an ABO bloodgroup system. In contrast, U.S. Pat. No. 5,639,611 discloses the use ofallele-specific amplification associated with the detection of a pointmutation that causes sickle cell anemia. However, genevariation-specific amplification or allele-specific amplification isproblematic in that it has low selectivity, and thus a more complicatedand time- and cost-intensive optimizing step is needed.

Such a method for detecting sequence variations, polymorphisms andmainly point mutations requires allele-specific amplification (or genevariation-specific amplification) particularly when a sequence variationto be detected is deficient compared to dominant variations in the samenucleic acid fragment (or the same gene).

For example, this situation occurs when sporadic tumor cells aredetected in the body fluid such as blood, serum or plasma by genevariation-specific amplification (U.S. Pat. No. 5,496,699). To this end,DNA is first isolated from the body fluid such as blood, serum orplasma, and DNA is derived from deficient, sporadic tumor cells andexcessive non-proliferative cells. Thus, mutations that are significantto tumor DNA in the k-ras gene should be detected from several copies inthe presence of an excessive amount of wild-type DNA.

All methods for gene variation-specific amplification disclosed in theprior art have the disadvantage that a 3′-terminal differentialoligonucleotide residue should be used. In addition, despite the use ofa 3′-differential nucleotide residue, these methods have thedisadvantage that primer extension occurs at low levels in the presenceof a suitable DNA polymerase even when a target nucleic acid is notexactly matched with a sequence variant to be detected. Particularly,when a specific sequence variant is detected by an excessive backgroundnucleic acid including a different sequence variant, it leads to a falsepositive result. The main reason for the disadvantage of the PCR-basedmethod is the incompatibility of a polymerase used in the method forsufficiently differentiating mismatched bases. Therefore, it is not yetpossible to directly obtain clear data on the presence or absence of amutation by PCR. To date, additional time- and cost-intensivepurification and analysis methods have been required for the cleardiagnosis of mutations. Therefore, a novel method for improving theselectivity of gene variation- or allele-specific PCR amplification willgreatly affect the reliability and robustness of direct gene variationor SNP analysis by PCR.

Therefore, there are continuous demands for the development of a DNApolymerase with increased gene variation specificity and an optimalreaction buffer in which various materials are mixed to exhibit a properfunction of the DNA polymerase.

The inventors had made efforts to develop a novel DNA polymerase thatcan improve the selectivity of gene variation-specific PCR amplificationand a reaction buffer for increasing its activity, confirming that genevariation specificity significantly increased when a mutation occurs atan amino acid residue at a specific position of Taq polymerase, and theactivity of the DNA polymerase with increased gene variation specificityincreases when the concentration of KCl, (NH₄)₂SO₄ and/or tetra methylammonium chloride (TMAC) among the components of the PCR buffercomposition is adjusted, and thus the present invention was completed.

DISCLOSURE Technical Problem

The present invention is directed to providing a DNA polymerase fordetecting one or more gene variations or SNPs in a target sequencehaving a gene variation or SNP.

The present invention is also directed to providing a nucleic acidsequence encoding the DNA polymerase according to the present invention,a vector including the nucleic acid sequence, and a host celltransformed with the vector.

The present invention is also directed to providing a method ofpreparing the DNA polymerase according to the present invention.

The present invention is also directed to providing a method of in vitrodetecting one or more gene variations or SNPs in one or more templatesusing the DNA polymerase of the present invention.

The present invention is also directed to providing a composition fordetecting a gene variation or SNP, which includes the DNA polymerase ofthe present invention.

The present invention is also directed to providing a kit for detectingthe DNA polymerase of the present invention, which includes thecomposition for detecting a gene variation or SNP according to thepresent invention.

The present invention is also directed to providing a PCR buffercomposition for increasing the activity of a DNA polymerase withincreased gene variation specificity.

The present invention is also directed to providing a PCR kit fordetecting a gene variation or SNP, which includes the PCR buffercomposition and/or the DNA polymerase with increased gene variationspecificity according to the present invention.

The present invention is also directed to providing a method of in vitrodetecting one or more gene variations or SNPs in one or more templatesusing the PCR kit according to the present invention.

Technical Solution

One aspect of the present invention provides a DNA polymerase comprisinga Taq polymerase amino acid sequence of SEQ ID NO: 1, the DNA polymeraseincluding

-   -   (a) a substitution at amino acid residue 507 in the amino acid        sequence of SEQ ID NO: 1; and    -   (b) (i) a substitution at amino acid residue 536 in the amino        acid sequence of SEQ ID NO: 1,    -   (ii) a substitution at amino acid residue 660 in the amino acid        sequence of SEQ ID NO: 1,    -   (iii) substitutions at amino acid residues 536 and 660 in the        amino acid sequence of SEQ ID NO: 1, or    -   (iv) substitutions at amino acid residues 536, 587 and 660 in        the amino acid sequence of SEQ ID NO: 1.

According to an exemplary embodiment of the present invention, thesubstitution at the amino acid residue 507 may be a substitution ofglutamic acid (E) with lysine (K), the substitution at the amino acidresidue 536 is a substitution of arginine (R) with lysine (K), thesubstitution at the amino acid residue 587 is a substitution of arginine(R) with isoleucine (I), and the substitution at the amino acid residue660 is a substitution of arginine (R) with valine (V).

According to another exemplary embodiment of the present invention, theDNA polymerase may discriminate a matched primer from a mismatchedprimer, wherein the matched primer may be hybridized with the targetsequence, and the mismatched primer may have a non-canonical nucleotideat the 3′ end thereof with respect to the hybridized target sequence.

According to still another exemplary embodiment of the presentinvention, the DNA polymerase may exhibit a Ct value lower than theamplification of the target sequence comprising the mismatched primer.

Another aspect of the present invention provides a nucleic acid sequenceencoding the DNA polymerase according to the present invention, a vectorincluding the nucleic acid sequence, and a host cell transformed withthe vector.

Still another aspect of the present invention provides a method ofpreparing a DNA polymerase, which includes: culturing the host cells;and isolating a DNA polymerase from the cell culture and a supernatantthereof.

Yet another aspect of the present invention provides a method of invitro detecting one or more gene variations or SNPs in one or moretemplates, the method including:

-   -   bringing the DNA polymerase according to the present invention        into contact with a) one or more templates;    -   b) one or more matched primers, one or more mismatched primers        or both of one or more matched primers and one or more        mismatched primers; and    -   c) a nucleoside triphosphate,    -   wherein the one or more matched primers and the one or more        mismatched primers are hybridized with a target sequence, and        the mismatched primer has a non-canonical nucleotide at base        position 7 from the 3′ end thereof with respect to the        hybridized target sequence.

According to an exemplary embodiment of the present invention, themethod may include a melting point analysis using a doublestrand-specific dye.

According to another exemplary embodiment of the present invention, themethod may be accomplished by real-time PCR, the analysis on agarose gelafter standard PCR, gene variation-specific amplification orallele-specific amplification through real-time PCR, tetra-primeramplification-refractory mutation system PCR or isothermalamplification.

Yet another aspect of the present invention provides a composition fordetecting a gene variation or SNP, comprising the DNA polymeraseaccording to the present invention.

Yet another aspect of the present invention provides a PCR kit includingthe composition for detecting a gene variation or SNP.

According to one exemplary embodiment of the present invention, the PCRkit may be used in competitive allele-specific TaqMan PCR (cast PCR),droplet digital PCR or MassARRAY.

According to another exemplary embodiment of the present invention, thePCR kit may further include one or more matched primers, one or moremismatched primers or both of one or more matched primers and one ormore mismatched primers, where the one or more matched primers and theone or more mismatched primers may be hybridized with a target sequence,and the mismatched primer may have a non-canonical nucleotide at baseposition 7 from the 3′ end thereof with respect to the hybridized targetsequence.

According to still another exemplary embodiment of the presentinvention, the PCR kit may further include a nucleoside triphosphate.

According to yet another exemplary embodiment of the present invention,the PCR kit may further include

-   -   a) one or more buffers;    -   b) a quantification reagent binding to double-stranded DNA;    -   c) a polymerase blocking antibody;    -   d) one or more control values or control sequences; and    -   e) one or more templates.

Yet another aspect of the present invention provides a PCR buffercomposition for increasing the activity of a DNA polymerase withincreased gene variation specificity, which includes 25 to 100 mM KCl;and 1 to 15 mM (NH₄)₂SO₄, wherein the final pH is 8.0 to 9.0.

According to one exemplary embodiment of the present invention, the KClconcentration may be 60 to 90 mM.

According to another exemplary embodiment of the present invention, the(NH₄)₂SO₄ concentration may be 2 to 8 mM.

According to still another exemplary embodiment of the presentinvention, the KCl concentration may be 70 to 80 mM, and the (NIH4)₂SO₄concentration may be 4 to 6 mM.

Yet another aspect of the present invention provides a PCR buffercomposition for increasing the activity of a DNA polymerase withincreased gene variation specificity, which includes 5 to 80 mM TMAC inthe above-described PCR buffer composition.

According to one exemplary embodiment of the present invention, the KClconcentration may be 40 to 90 mM.

According to another exemplary embodiment of the present invention, the(NH₄)₂SO₄ concentration may be 1 to 7 mM.

According to still another exemplary embodiment of the presentinvention, the TMAC concentration may be 15 to 70 mM, the KClconcentration may be 50 to 80 mM, and the (NH₄)₂SO₄ concentration may be1.5 to 6 mM.

According to yet another exemplary embodiment of the present invention,the PCR buffer composition may further include Tris·Cl and MgCl₂.

Yet another aspect of the present invention provides a PCR kit fordetecting a gene variation or SNP, which includes the above-describedPCR buffer composition.

According to one exemplary embodiment of the present invention, the PCRkit may include a DNA polymerase comprising a Taq polymerase amino acidsequence of SEQ ID NO: 1, where the DNA polymerase has thesubstitution(s) of the following amino acids:

-   -   (a) a substitution at amino acid residue 507 in the amino acid        sequence of SEQ ID NO: 1; and    -   (b) (i) a substitution at amino acid residue 536 in the amino        acid sequence of SEQ ID NO: 1,    -   (ii) a substitution at amino acid residue 660 in the amino acid        sequence of SEQ ID NO: 1,    -   (iii) substitutions at amino acid residues 536 and 660 in the        amino acid sequence of SEQ ID NO: 1, or    -   (iv) substitutions at amino acid residues 536, 587 and 660 in        the amino acid sequence of SEQ ID NO: 1.

According to another exemplary embodiment of the present invention, thesubstitution at the amino acid residue 507 may be a substitution ofglutamic acid (E) with lysine (K), the substitution at the amino acidresidue 536 may be a substitution of arginine (R) with lysine (K), thesubstitution at the amino acid residue 587 may be a substitution ofarginine (R) with isoleucine (I), and the substitution at the amino acidresidue 660 may be a substitution of arginine (R) with valine (V).

According to still another exemplary embodiment of the presentinvention, the PCR kit may further include a) a nucleoside triphosphate;b) a quantification reagent binding to double-stranded DNA; c) apolymerase blocking antibody, d) one or more control values or controlsequences; and e) one or more templates.

Yet another aspect of the present invention provides a method of invitro detecting one or more gene variations or SNPs in one or moretemplates using the PCR kit of the present invention.

Advantageous Effects

Since the DNA polymerase with increased gene variation specificityaccording to the present invention has a higher mismatch-to-matchextension selectivity than conventional Taq polymerase, reliable genevariation-specific amplification is possible without any substratemodification. The present invention also provides an optimal PCR buffercomposition that allows the proper function of a DNA polymerase withincreased gene variation specificity to be effectively exhibited, andreliable gene variation-specific amplification is possible byconsiderably increasing the activity of the DNA polymerase using the DNApolymerase with increased gene variation specificity. Moreover, a kitincluding a PCR buffer composition and/or the DNA polymerase withincreased gene variation specificity according to the present inventioncan effectively detect a gene variation or SNP, and thus can be usefullyapplied to the medical diagnosis of a disease and recombinant DNAstudies.

DESCRIPTION OF DRAWINGS

FIG. 1 shows a process of preparing Taq DNA polymerases having R536K,R660V and R536K/R660V variations: (a) the schematic representation offragment PCR and overlap PCR; (b) the result of electrophoresis foramplified products obtained by the fragment PCR; and (c) the result ofelectrophoresis for amplified products obtained by full-lengthamplification through overlap PCR.

FIG. 2 shows the result of electrophoresis for a pUC19 vector which isdigested with restriction enzymes EcoRI/XbaI and treated with SAP andthe purified overlap PCR products of (c) in FIG. 1 .

FIG. 3 is the schematic representation of fragment PCR and overlap PCRduring the preparation of Taq DNA polymerases having E507K, E507K/R536K,E507K/R660V and E507K/R536K/R660V variations, respectively.

FIG. 4 shows the result of electrophoresis for a pUC19 vector digestedwith EcoRI/XbaI and then treated with SAP and the purified overlap PCRproduct of FIG. 3 for gel extraction.

FIG. 5 is the schematic representation of the process of preparing a PCRtemplate by collecting oral epithelial cells.

FIGS. 6 a to 6 d show the results of AS-qPCR for rs1408799 using Taqpolymerases having E507K/R536K, E507K/R660V and E507K/R536K/R660Vvariations according to the present invention, and Taq polymerase havingan E507K variation is used as a control.

FIGS. 7 a to 7 d show the results of AS-qPCR for rs1015362 using Taqpolymerases having E507K/R536K, E507K/R660V and E507K/R536K/R660Vvariations according to the present invention, and Taq polymerase havingan E507K variation is used as a control.

FIGS. 8 a to 8 d shows the results of AS-qPCR for rs4911414 using Taqpolymerases having E507K/R536K, E507K/R660V and E507K/R536K/R660Vvariations according to the present invention, and Taq polymerase havingan E507K variation is used as a control.

FIG. 9 shows the process of preparing Taq DNA polymerase havingE507K/R536K/R587I/R660V variations: (a) the schematic representation offragment PCR and overlap PCR; and (b) the result of electrophoresis forthe amplified product obtained by the fragment PCR.

FIGS. 10 a to 10 d show the results of AS-qPCR for a template having anSNP of Q61H in a KRAS gene using a E507K/R536K/R587I/R660V polymerase,wherein FIGS. 10 a and 10 b show the results obtained using a 24-merlong primer, FIGS. 10 c and 10 d show the results obtained using a18-mer long primer, and Taq polymerase having E507K/R536K/R660Vvariations is used as a control.

FIG. 11 shows the result obtained by AS-qPCR for a template having anSNP of G13D in a KRAS gene using a E507K/R536K/R587I/R660V polymerase,and Taq polymerase having E507K/R536K/R660V variations is used as acontrol.

FIG. 12 shows the result obtained by AS-qPCR for a template having anSNP of G12S in a KRAS gene using a E507K/R536K/R587I/R660V polymerase,and Taq polymerase having E507K/R536K/R660V variations is used as acontrol.

FIG. 13 shows the result obtained by AS-qPCR for a template having anSNP of L585R in an EGFR gene using a E507K/R536K/R587I/R660V polymerase,and Taq polymerase having E507K/R536K/R660V variations is used as acontrol.

FIGS. 14 a to 14 d are graphs showing the amplification delay effect bymismatch according to the change in KCl concentration of a reactionbuffer using E507K, E507K/R536K, E507K/R660V and E507K/R536K/R660V Taqpolymerases of the present invention.

FIG. 15 shows the result of electrophoresis for a PCR product obtainedby amplification with a constantly fixed (NH₄)₂SO₄ concentration andvarious KCl concentrations to confirm the optimal KCl concentration in areaction buffer.

FIG. 16 shows the result of electrophoresis for a PCR product obtainedby amplification with a constantly fixed KCl concentration and various(NH₄)₂SO₄ concentrations to confirm the optimal (NH₄)₂SO₄ concentrationin a reaction buffer.

FIG. 17 is the graph showing the amplification delay effect by mismatchaccording to the change in (NH₄)₂SO₄ concentration in a reaction buffer.

FIGS. 18 a and 18 b are graphs showing the amplification delay effect bymismatch according to the change in TMAC concentration after KCl and(NH₄)₂SO₄ concentrations are constantly fixed in a reaction buffer.

FIGS. 19 a and 19 b are graphs showing the amplification delay effect bymismatch according to the change in KCl concentration after TMAC and(NH₄)₂SO₄ concentrations are constantly fixed in a reaction buffer.

MODES OF THE INVENTION

Hereinafter, the present invention will be described in further detail.

As described above, to improve the disadvantages of the genevariation-specific amplification method disclosed in the conventionalart, there is a continuous demand for the development of a DNApolymerase with increased gene variation specificity and an optimalreaction buffer in which various materials are mixed such that the DNApolymerase can exhibit the proper function, and the development of sucha method greatly affects the reliability and robustness of direct genevariation or SNP analysis by PCR. The inventors had made an effort todevelop a novel DNA polymerase capable of improving the selectivity ofgene variation-specific PCR amplification and a reaction buffer forincreasing its activity, confirming that gene variation specificitysignificantly increased when a mutation occurs on an amino acid residueat a specific position of Taq polymerase, and the activity of the DNApolymerase with increased gene variation-specific amplificationefficiency increases when the concentration of KCl, (NH₄)₂SO₄ and/ortetra methyl ammonium chloride (TMAC) among the components of the PCRbuffer composition is adjusted, and thus the present invention wascompleted.

Since the DNA polymerase with increased gene variation specificityaccording to the present invention has a higher mismatch-to-matchextension selectivity than conventional Taq polymerase, reliable genevariation-specific amplification is possible without any substratemodification. The present invention also provides an optimal PCR buffercomposition that allows the proper function of a DNA polymerase withincreased gene variation specificity to be effectively exhibited, andreliable gene variation-specific amplification is possible byconsiderably increasing the activity of the DNA polymerase using the DNApolymerase with increased gene variation specificity. Moreover, a kitincluding a PCR buffer composition and/or the DNA polymerase withincreased gene variation specificity according to the present inventioncan effectively detect a gene variation or SNP, and thus can be usefullyapplied to the medical diagnosis of a disease and recombinant DNAstudies.

Hereinafter, terms used herein will be defined.

The “amino acid” refers to any monomer unit that can be incorporatedinto a peptide, a polypeptide or a protein. The term “amino acid” usedherein includes 20 natural or genetically encoded alpha-amino acids asfollows: alanine (Ala or A), arginine (Arg or R), asparagine (Asn or N),aspartic acid (Asp or D), cysteine (Cys or C), glutamine (Gln or Q),glutamic acid (Glu or E), glycine (Gly or G), histidine (His or H),isoleucine (Ile or 1), leucine (Leu or L), lysine (Lys or K), methionine(Met or M), phenylalanine (Phe or F), proline (Pro or P), serine (Ser orS), threonine (Thr or T), tryptophan (Trp or W), tyrosine (Tyr or Y),and valine (Val or V).

Amino acids are typically organic acids, which substituted orunsubstituted amino groups, substituted or unsubstituted carboxylgroups, and one or more side chains or groups, or any analogs of thesegroups. Exemplary side chains include, for example, thiol, seleno,sulfonyl, alkyl, aryl, acyl, keto, azido, hydroxyl, hydrazine, cyano,halo, hydrazide, alkenyl, alkynyl, ether, borate, boronate, phospho,phosphono, phosphine, heterocyclic, enone, imine, aldehyde, ester,thioacid, hydroxylamine, or any combination thereof.

Other exemplary amino acids include the following amino acids, but thepresent invention is not limited thereto: an amino acid including aphotoactivatable crosslinking agent, a metal-binding amino acid, aspin-labeled amino acid, a fluorescent amino acid, a metal-containingamino acid, a novel functional group-containing amino acid, an aminoacid covalently or non-covalently interacting with another molecule, aphotocaged and/or photoisomerizable amino acid, a radioactive aminoacid, an amino acid including a biotin or biotin analog, a glycosylatedamino acid, an amino acid modified with another carbohydrate, an aminoacid including polyethylene glycol or polyether, a heavyatom-substituted amino acid, chemodegradable and/or photodegradableamino acid(s), a carbon-linked sugar-containing amino acid, aredox-active amino acid, an amino thioacid-containing amino acid, and anamino acid including one or more toxic parts.

Regarding the DNA polymerase of the present invention, the term “mutant”means a recombinant polypeptide including one or more amino acidsubstitutions, compared to a corresponding naturally-occurring orunmodified DNA polymerase.

The term “thermostable polymerase (referring to a thermostable enzyme)”has thermal resistance, has sufficient activity to achieve subsequentpolynucleotide extension, and is not irreversibly denatured(inactivated) when treated at elevated temperatures for the timerequired to achieve the denaturation of a double-stranded nucleic acid.As used herein, it is suitable for a reaction such as PCR to be used ata cycling temperature. Herein, irreversible denaturation refers to thepermanent and complete loss of enzyme activity. The enzyme activity ofthe thermostable polymerase refers to the catalysis of a nucleotidecombination by a method suitable for the formation of a polynucleotideextension product which is complementary to a template nucleic acidstrand. Thermophilic bacteria-derived thermostable DNA polymerasesinclude, for example, DNA polymerases derived from Hermotoga maritima,Thermus aquaticus, Thermus thermophilus, Thermus flabus,Thermodipyliporphis, Thermus sp. Sps17, Thermus sp. Z05, Thermuscaldophilus, Bacillus caldotenax, Thermotoga neopolitanica andThermosipo africanus.

The term “thermoactive” refers to an enzyme maintaining a catalyticproperty at temperatures (i.e., 45 to 80° C.) conventionally used inreverse transcription or annealing/extension steps in RT-PCR and/or PCRreactions. The thermostable enzyme is not irreversibly inactivated ordenatured when treated at elevated temperatures required for nucleicacid denaturation. The thermoactive enzyme may be thermostable or maynot thermostable. The thermoactive DNA polymerase may include, but notlimited to, DNA or RNA dependent on thermophilic or mesophilic species.

The term “host cell” includes single-cellular prokaryotic and eukaryoticorganisms (e.g., bacteria, yeast, and actinomycetes) and single cellsderived from higher plant, an animal, or both thereof.

The term “vector” refers to a DNA molecule which is replicable and ableto deliver foreign DNA such as a gene to a recipient cell, for example,a plasmid, a phage, or an artificial chromosome. The “plasmid,”“vector,” or “plasmid vector” used herein may be used interchangeably.

The term “nucleotide” may be a deoxyribonucleic acid (DNA) or aribonucleic acid (RNA), which is present in a single strand or doublestrand, and unless particularly described otherwise, an analog of anatural nucleotide may be included.

The term “nucleic acid” or “polynucleotide” refers to a DNA or RNApolymer, or a polymer that can correspond to an analog thereof. Thenucleic acid may be, for example, a chromosome or chromosome fragment, avector (e.g., an expression vector), an expression cassette, a naked DNAor RNA polymer, a product of a polymerase chain reaction (PCR), anoligonucleotide, a probe, or a primer, but the present invention is notlimited thereto. The nucleic acid may be, for example, asingle-stranded, double-stranded, or triple-stranded, but is not limitedto any specific length. Unless particularly defined otherwise, aspecific nucleic acid sequence includes a complementary sequence inaddition to a random sequence noted herein, or encodes the same.

The “primer” refers to a polynucleotide that can serve as a startingpoint of nucleic acid synthesis in a template-direction under theconditions for the initiation of the extension of a polynucleotide.Primers may also be used in the process of synthesis mediated by variousother oligonucleotides which are included as initiators of de novo RNAsynthesis and an in vitro transcription-related process. Primers aretypically single-stranded oligonucleotides (e.g.,oligodeoxyribonucleotides). The suitable length of a primer variestypically in the range from 6 to 40 nucleotides, and more typically, 15to 35 nucleotides, according to the intended use. A short primermolecule generally requires a lower temperature to form a sufficientlystable hybridization complex with a template. A primer is not requiredto correspond to the exact sequence of a template, but needs to besufficiently complementary to be hybridized with the template subject toextension. In a specific exemplary embodiment, the term “primer pair”means a primer set comprising a 5′-sense primer which is complementarilyhybridized to the 5′ end of a nucleic acid sequence to be amplified, anda 3′-antisense primer which is hybridized to the 3′ end of the sequenceto be amplified. A primer may be labeled, if necessary, by being mixedwith a marker to be detected by spectroscopic, photochemical,biochemical, immunochemical or chemical means. For example, a usefulmarker is as follows: 32P, a fluorescent dye, an electron-dense reagent,an enzyme (conventionally used in ELISA), biotin, or a protein that canbe used with hapten and an anti-serum or monoclonal antibody.

The term “5′-nuclease probe” refers to an oligonucleotide having one ormore luminescent markers which are used in a 5′-nuclease reaction fortargeting nucleic acid detection. In some exemplary embodiments, forexample, a 5′-nuclease probe only has a single luminescent part (e.g., afluorescent dye or the like). In a specific exemplary embodiment, a5-nuclease probe has a self-complementary region to form a hairpinstructure under selective conditions. In some exemplary embodiments, a5′-nuclease probe has two or more markers, and one of the two markers isseparated or degraded from the oligonucleotide and then released with anincreased radiation intensity. In a specific exemplary embodiment, a5′-nuclease probe is labeled with two different fluorescent dyes, forexample, a 5′-end reporter dye and a 3′-end quencher dye. In someexemplary embodiments, a 5′-nuclease probe is labeled at one or morepositions in addition to or other than the ends. When the probe isintact, typically, energy transfer occurs between two fluorescentmaterials to partially or completely quench fluorescence emitted from areporter dye. During extension in PCR, for example, a 5′-nuclease probebinding to a template nucleic acid is degraded by the activity of nolonger quenching the fluorescence emission of a reporter dye, forexample, the 5′ or 3′-nuclease activity of Taq polymerase or a differentpolymerase. In some exemplary embodiments, a 5′-nuclease probe may belabeled with two or more different reporter dyes and a 3′-end quencherdye or a part thereof.

The term “FRET” or “fluorescence resonance energy transfer” or “FoersterResonance Energy Transfer” refers to the transfer of energy between twoor more chromophores, donor chromophores and recipient chromophores(referred to as quenchers). Typically, when a donor is excited byradiating light with an appropriate wavelength, energy is transferred toa recipient. The recipient typically re-radiates energy transferred inthe form of light radiated with a different wavelength. When therecipient is a “dark” quencher, it disperses energy transferred in aform other than light. Whether a specific fluorescent material serves asa donor or recipient is dependent on the properties of other members ofthe FRET pair. Conventionally used donor-recipient pairs include aFAM-TAMRA pair. Conventionally used quenchers are DABCYL and TAMRA.Conventionally used dark quenchers are as follows: BlackHole Quenchers(BHQ), (Biosearch Technologies, Inc., Novato, Cal.), Iowa Black(Integrated DNA Tech., Inc., Coralville, Iowa), and BlackBerry Quencher650 (BBQ-650) (Berry & Assoc., Dexter, Mich.).

The term “conventional” or “natural” used to describe a nucleic acidbase, a nucleoside triphosphate or a nucleotide refers to thosenaturally occurring in the polynucleotides described herein (i.e., forDNA, dATP, dGTP, dCTP and dTTP). In addition, dTTP and 7-deaza-dGTP arefrequently used instead of dGTP, and may be used instead of dATP in anin vitro DNA synthesis reaction such as sequencing.

The term “unconventional” or “modified” used to describe a nucleic acidbase, a nucleoside triphosphate or a nucleotide refers to themodification, derivative or analog of a conventional base, nucleoside ornucleotide, which naturally occurs in a specific polynucleotide. Aspecific, unconventional nucleotide is modified at the 2′ position ofthe ribose, compared with conventional dNTP. Therefore, although anucleotide naturally occurring in RNA is a ribonucleotide (i.e., ATP,GTP, CTP, UTP, and collectively, rNTP), since the nucleotide has ahydroxyl group at the 2′ position of the sugar, compared with dNTPhaving no hydroxyl group, as used herein, the ribonucleotide is anucleotide which is not conventionally used as a substrate for a DNApolymerase. As used herein, an unconventional nucleotide includes acompound used as a terminator for nucleic acid sequencing, but thepresent invention is not limited thereto. An exemplary terminatorcompound includes a compound having a 2′,3′-dideoxy structure, but thepresent invention is not limited thereto, and is referred to as adideoxynucleoside triphosphate. Dideoxynucleoside triphosphates such asddATP, ddTTP, ddCTP and ddGTP are collectively referred to as ddNTP.Additional examples of terminator compounds include 2′-PO4 analogs of aribonucleotide. Other unconventional nucleotides includephosphorothioate dNTP ([[α]-S]dNTP), 5′-[α]-borano-dNTP,[α]-methyl-phosphonate dNTP, and ribonucleoside triphosphate (rNTP). Anunconventional base may be labeled with a radioactive isotope, such as32P, 33P, or 35S; a fluorescent marker, a chemoluminescent marker, abioluminescent marker, a hapten marker such as biotin; or an enzymemarker such as streptavidin or avidin. A fluorescent marker may be anegatively-charged dye such as a fluorescein-family dye, or aneutrally-charged dye such as a rhodamine-family dye, or apositively-charged dye such as a cyanine-family dye. Fluorescein-familydyes include, for example, FAM, HEX, TET, JOE, NAN and ZOE.Rhodamine-family dyes include Texas Red, ROX, R110, R6G, and TAMRA.Various dyes or nucleotides labeled with FAM, HEX, TET, JOE, NAN, ZOE,ROX, R110. R6G, Texas Red and TAMRA are commercially available fromPerkin-Elmer (Boston, MA), Applied Biosystems (Foster City, CA), orInvitrogen/Molecular Probes (Eugene, OR). Cyanine-family dyes includeCy2, Cy3, Cy5 and Cy7, and are commercially available from GE HealthcareUK Limited (Amersham Place, Little Chalfont, Buckinghamshire, England).

The term “mismatch discrimination” refers to the ability of abiocatalyst (e.g., an enzyme such as a polymerase, ligase, or the like)to discriminate a fully-complementary sequence from amismatch-containing sequence when a nucleic acid (e.g., primer or adifferent oligonucleotide) is extended by attaching (for example,covalently) one or more nucleotides to the nucleic acid in atemplate-dependent manner. The term “mismatch discrimination” refers tothe ability of a biocatalyst to discriminate a fully-complementarysequence from a mismatch-containing (approximately complementary)sequence, that is, an extended nucleic acid (e.g., a primer or differentoligonucleotide) has a mismatch in the 3′-end nucleic acid, comparedwith a nucleic acid-hybridized template. In some exemplary embodiments,an extended nucleic acid includes a mismatch at the 3′ end with respectto a fully-complementary sequence. In some exemplary embodiments, anextended nucleic acid includes a mismatch at the penultimate (N-1) 3′position and/or at the N-2 position relative to the fully complementarysequence.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meanings as generally understood by those of ordinaryskill in the art.

The present invention relates to a DNA polymerase comprising a Taqpolymerase amino acid sequence of SEQ ID NO: 1, the DNA polymeraseincluding

-   -   (a) a substitution at amino acid residue 507 in the amino acid        sequence of SEQ ID NO: 1;    -   (b) (i) a substitution at amino acid residue 536 in the amino        acid sequence of SEQ ID NO: 1,    -   (ii) a substitution at amino acid residue 660 in the amino acid        sequence of SEQ ID NO: 1,    -   (iii) substitutions at amino acid residues 536 and 660 in the        amino acid sequence of SEQ ID NO: 1, or    -   (iv) substitutions at amino acid residues 536, 587 and 660 in        the amino acid sequence of SEQ ID NO: 1.

The “Taq polymerase” is a heat-resistant DNA polymerase named afterthermophilic bacteria Thermus aquaticus, and was first isolated from thebacteria. Thermus aquaticus are bacteria living in hot springs andhydrothermal vents, and the Taq polymerase is an enzyme that cantolerate a protein-denaturing condition (high temperature) requiredduring PCR. The Taq polymerase has an optimal activity temperature of 75to 80° C., has a half-life of 2 hours or more at 92.5° C., 40 minutes at95° C. and 9 minutes at 97.5° C., and can replicate 1000-bp DNA within10 seconds at 72° C. It lacks 3′-5′ exonuclease proofreading activity,resulting in an error rate of approximately 1 in 9,000 nucleotides. Forexample, when heat-resistant Taq is used, PCR may be performed at a hightemperature (60° C. or more). The amino acid sequence set forth in SEQID NO: 1 is used as a reference sequence for the Tag polymerase.

According to an exemplary embodiment of the present invention, thesubstitution at amino acid residue 507 is a substitution of glutamicacid (E) with lysine (K), the substitution at amino acid residue 536 isa substitution of arginine (R) with lysine (K), the substitution atamino acid residue 587 is a substitution of arginine (R) with isoleucine(I), and the substitution at amino acid residue 660 may be asubstitution of arginine (R) with valine (V).

In the present invention, the Taq polymerase in which glutamic acid (E)is substituted with lysine (K) at the amino acid residue 507 in theamino acid sequence of SEQ ID NO: 1 is named “E507K” (SEQ ID NO: 2); theTaq polymerase in which glutamic acid (E) is substituted with lysine (K)at the amino acid residue 507, and arginine (R) is substituted withlysine (K) at the amino acid residue 536 in the amino acid sequence ofSEQ ID NO: 1 is named “E507K/R536K” (SEQ ID NO: 6); the Taq polymerasein which glutamic acid (E) is substituted with lysine (K) at the aminoacid residue 507, and arginine (R) is substituted with valine (V) at theamino acid residue 660 in the amino acid sequence of SEQ ID NO: 1 isnamed “E507K/R660V” (SEQ ID NO: 7); the Taq polymerase in which glutamicacid (E) is substituted with lysine (K) at the amino acid residue 507,arginine (R) is substituted with lysine (K) at the amino acid residue536, and arginine (R) is substituted with valine (V) at the amino acidresidue 660 in the amino acid sequence of SEQ ID NO: 1 is named“E507K/R536K/R660V” (SEQ ID NO: 8); and finally the Taq polymerase inwhich glutamic acid (E) is substituted with lysine (K) at the amino acidresidue 507, arginine (R) is substituted with lysine (K) at the aminoacid residue 536, arginine (R) is substituted with isoleucine (I) at theamino acid residue 587, and arginine (R) is substituted with valine (V)at the amino acid residue 660 in the amino acid sequence of SEQ ID NO: 1is named “E507K/R536K/R587I/R660V” (SEQ ID NO: 37).

According to an exemplary embodiment of the present invention, the DNApolymerase discriminates a matched primer from a mismatched primer, thematched primer and the mismatched primer are hybridized with a targetsequence, and the mismatched primer may include a non-canonicalnucleotide at the 3′ end with respect to a hybridized target sequence.

The mismatched primer is a hybrid oligonucleotide which should besufficiently complementary to be hybridized with the target sequence,but does not correspond to the exact sequence of the target sequence.

The “canonical nucleotide” or “complementary nucleotide” means astandard Watson-Crick base pair, A-U, A-T or G-C.

The “non-canonical nucleotide” or “non-complementary nucleotide” meansA-C, A-G, G-U, G-T, T-C, T-U, A-A, G-G, T-T, U-U, C-C, or C-U other thanthe Watson-Crick base pairs.

According to an exemplary embodiment of the present invention, with theDNA polymerase, the amplification of a target sequence including amatched primer may exhibit a lower Ct value than the amplification of atarget sequence including a mismatched primer.

For example, the DNA polymerase may allow one or more nucleotides tocovalently bind to a primer, thereby extending a matched primer withgreater efficiency than a mismatched primer in a targetsequence-dependent manner. Here, greater efficiency may be observed at alower Ct value for a matched primer, compared with the mismatchedprimer, for example, in RT-PCR. The difference in Ct value between thematched primer and the mismatched primer may be 10 or more, andpreferably, 10 to 20, or there may be no synthesis of an amplicon by amismatched primer.

For example, such a difference means that a product formed by standardPCR using a forward primer and a reverse primer, which are matched in afirst reaction, and a reverse primer matched with a mismatched forwardprimer in a second reaction with the same experiment settings is largerin the first reaction than in the second reaction.

A Ct (threshold crossing cycle) value represents a DNA quantificationmethod by quantitative PCR, which depends on plotting the fluorescencerepresenting the number of cycles on a log scale. The threshold forDNA-based fluorescence detection is set slightly higher than the minimumbackground. The number of cycles required for fluorescence to cross thethreshold is called Ct or a quantification cycle (Cq) following the MIQEguidelines. The Ct value for the given reaction is defined as the numberof cycles required for fluorescence emission to cross a fixed threshold.For example, SYBR Green I and a fluorescent probe may be used inreal-time PCR for template DNA quantification. Fluorescence emitted froma sample is collected every cycle during PCR, and plotted against thenumber of cycles. A starting template concentration is inverselyproportional to the time at which the fluorescent signal is first shown.The signal appears earlier as a template concentration is higher (shownat a low number of cycles).

The present invention also relates to a nucleic acid sequence encodingthe above-described DNA polymerase, and a vector and a host cell, whichinclude the nucleic acid sequence. Various vectors may be prepared usingthe nucleic acid encoding the DNA polymerase of the present invention.Any vector having a replicon and a control sequence, which are derivedfrom a species compatible with a host cell, may be used. The vector ofthe present invention may be an expression vector, and has nucleic acidregions for regulating transcription and translation, which are operablylinked to the nucleic acid sequence encoding the DNA polymerase of thepresent invention. The regulatory sequence refers to a DNA sequencerequired for the expression of a coding sequence operably linked to aspecific host organism. For example, a control sequence suitable for aprokaryote includes a promoter, any operating sequence and a ribosomebinding sequence. In addition, a vector may include a “positiveretroregulatory element (PRE)” to increase the half-life of mRNA to betranscribed. Transcription and translation regulatory nucleic acidregions may be generally suitable for host cells used to express apolymerase. Various types of suitable expression vectors and regulatorysequences are known to be used for various host cells. Generally,transcription and translation regulatory sequences may include, forexample, a promoter sequence, a ribosome binding site, transcriptioninitiation and termination sequences, translation initiation andtermination sequences, and an enhancer or activation sequence. In atypical, exemplary embodiment, regulatory sequences include a promoterand transcription initiation and termination sequences. Typically, avector also includes a polylinker region containing several restrictionsites for inserting foreign DNA. In a specific exemplary embodiment, the“fusion flag” is used to promote purification, and if necessary, atag/flag is subsequently removed (e.g., “His-Tag”). However, whenthermoactive and/or thermostable protein(s) is(are) purified frommesophilic hosts (e.g., E. coli) using a “heating step,” the fusionflags are generally unnecessary. A suitable vector containing a DNAencoding replication sequence, a regulatory sequence and a phenotypeselection gene is constructed, and a mutant polymerase of interest isprepared using a standard recombinant DNA technique. An isolatedplasmid, a viral vector and a DNA fragment are digested and cleaved, andthen ligated with each other in a specific order to form a desiredvector as known in the art.

In an exemplary embodiment of the present invention, an expressionvector contains a selectable marker gene to select a transformed hostcell. Selection genes are known in the art, and may vary according tothe host cells used herein. Suitable selection genes may include thegene coding for ampicillin and/or tetracycline resistance, and may allowcells in which these vectors are cultured in the presence of theseantibiotics to be transformed.

In an exemplary embodiment of the present invention, a nucleic acidsequence encoding the DNA polymerase of the present invention may beintroduced into cells alone or in combination with a vector. Theintroduction or equivalent expressions thereof refer to a nucleic acidentering cells in the method suitable for subsequent integration,amplification and/or expression. The introduction method includes, forexample, CaPO₄ precipitation, liposome fusion, LIPOFECTIN®,electrophoresis, and viral infection.

Prokaryotes are used as host cells in an early cloning step of thepresent invention. They are particularly useful for rapidly preparing agreat quantity of DNA, for preparing a single-stranded DNA template usedin site-directed mutagenesis, for simultaneously screening many mutants,and for DNA sequencing of generated mutants. Suitable prokaryotic hostcells include E. coli K12 strain 94 (ATCC No. 31,446), E. coli strainW3110 (ATCC No. 27,325), E. coli K12 strain DG116 (ATCC No. 53,606), E.coli X1776 (ATCC No. 31,537), and E. coli B; many other strains of E.coli, such as HB101, JM101, NM522, NM538 and NM539, and other speciessuch as Bacilli, e.g., Bacillus subtilis, other Enterobacteriaceae,e.g., Salmonella typhimurium or Serratia marcescens, and prokaryoticgenera including various Pseudomonas sp. may be used as hosts.Typically, plasmids used in the transformation of E. coli includepBR322, pUCI8, pUCI9, pUCI18, pUC119 and Bluescript M13. However, manyother suitable vectors may also be used.

The present invention also provides a method of preparing a DNApolymerase, which includes: culturing the host cells; and isolating aDNA polymerase from a cell culture and a supernatant thereof.

The DNA polymerase of the present invention is prepared by culturinghost cells transformed with an expression vector containing a nucleicacid sequence encoding the DNA polymerase under suitable conditionsinducing or causing the expression of the DNA polymerase. A method ofculturing the transformed host cells under conditions suitable forprotein expression is known in the art. Host cells suitable for thepreparation of a polymerase from a lambda (λ) μL promoter-containingplasmid vector include E. coli strain DG116 (ATCC No. 53606). Whenexpressed, the polymerase may be collected and isolated.

After purification, mismatch discrimination of the DNA polymerase of thepresent invention may be assayed. For example, mismatch discriminationactivity is measured by comparing the amplification of a target sequenceperfectly matched with a primer with respect to the amplification of atarget having a single base mismatch at the 3′ end of a primer. Theamplification may be detected in real time by using, for example, aTaqMan™ probe. The ability of a polymerase to distinguish between twotarget sequences may be assumed by comparing Cts in two reactions.

Therefore, the present invention provides a method of in vitro detectingone or more gene variations or SNPs in one or more templates, the methodincluding:

-   -   bringing the DNA polymerase according to the present invention        into contact with a) one or more templates,    -   b) a nucleoside triphosphate, and    -   c) one or more matched primers, one or more mismatched primers        or both of one or more matched primers and one or more        mismatched primers, wherein the one or more matched primers and        the one or more mismatched primers are hybridized with a target        sequence, and the mismatched primer has a non-canonical        nucleotide at base position 7 from the 3′ end thereof with        respect to the hybridized target sequence.

The “single-nucleotide polymorphism (SNP)” refers to a genetic change orvariation showing the difference of a single base (A, T, G or C) in aDNA base sequence.

In the method of in vitro detecting a gene variation or SNP, a targetsequence may be present in a test sample, including, for example, DNA,cDNA or RNA, and preferably, genomic DNA. The test sample may be a celllysate prepared from bacteria, a bacterial culture, or a cell culture.In addition, the test sample may be one included in an animal,preferably, a vertebrate, and more preferably, a human subject. Thetarget sequence may be genomic DNA, preferably, genomic DNA of anindividual, more preferably, bacteria or a vertebrate, and mostpreferably, genomic DNA of a human subject.

The SNP detection method of the present invention may include analysisof a melting temperature using a double strand-specific dye such as SYBRGreen I.

The analysis of a melting temperature curve may be performed in areal-time PCR instrument such as ABI 5700/7000 (96-well format) or ABI7900 (384-well format) instrument with onboard software (SDS 2.1).Alternatively, the analysis of a melting temperature curve may beperformed as end-point analysis.

The “dye binding to double-stranded DNA” or “double strand-specific dye”may be used when high fluorescence is emitted while binding todouble-stranded DNA, rather than in an unbound state. Examples of thesedyes are SOYTO-9, SOYTO-13, SOYTO-16, SOYTO-60, SOYTO-64, SOYTO-82,ethidium bromide (EtBr), SYTOX Orange, TO-PRO-1, SYBR Green I, TO-PRO-3or EvaGreen. These dyes excluding EtBr and EvaGreen (Qiagen) have beentested in real-time applications.

The method of in vitro detecting a gene variation or SNP may beperformed by real-time PCR, analysis on agarose gel after standard PCR,gene variation-specific amplification or allele-specific amplificationthrough real-time PCR, tetra-primer amplification-refractory mutationsystem PCR or isothermal amplification, but the present invention is notlimited thereto.

For example, the SNP detection method of the present invention may beperformed using sequencing, mini-sequencing, allele-specific PCR,dynamic allele-specific hybridization (DASH), a PCR extension assay(e.g., single base extension; SBE), PCR-SSCP, a PCR-RFLP assay or TaqManmethod, SNPlex platform (Applied Biosystems), mass spectrometry (e.g.,MassARRAY system of Sequenom), or a Bio-Plex system (BioRad).

The “standard PCR” is a technique for amplifying single or severalcopies of DNA or cDNA known to a technician of ordinary skill in theart. Almost all PCR techniques use a thermostable DNA polymerase such asTaq polymerase or Klen Taq. A DNA polymerase uses single-stranded DNA asa template, and enzymatically assembles a new DNA strand fromnucleotides using oligonucleotide primers. Amplicons generated by PCRmay be analyzed on, for example, agarose gel.

The “real-time PCR” may monitor a PCR process in real time. Therefore,data is collected throughout the PCR process, not at the end of PCR. Inthe real-time PCR, the reaction is characterized by the point of timeduring a cycle when amplification is first detected, rather than theamount of a target accumulated after a fixed number of cycles. Usually,both of dye-based detection and probe-based detection are used toperform quantitative PCR.

The “allele-specific amplification (ASA)” is an amplification techniquefor designing PCR primers to discriminate templates with differentsingle nucleotide residues.

The “allele-specific amplification or gene variation-specificamplification through real-time PCR” is a highly effective method fordetecting a gene variation or SNP. Unlike most of other methods fordetecting a gene variation or SNP, the pre-amplification of a targetgene material is not needed. ASA combines amplification and detection ina single reaction based on the discrimination between matched andmismatched primer/target sequence complex. The increase in amplified DNAduring the reaction may be monitored in real time with the increase influorescent signal caused by a dye such as SYBR Green I emitted uponbinding to double-stranded DNA. The allele-specific amplification orgene variation-specific amplification through real-time PCR shows thedelay or absence of a fluorescent signal when a primer is mismatched. Inthe gene variation or SNP detection, such amplification providesinformation on the presence or absence of a gene variation or SNP.

The “tetra-primer amplification-refractory mutation system PCR” isamplification of all of wild-type and mutant alleles with a controlfragment in single tube PCR. A non-allele-specific control amplicon isamplified by two common (outside) primers flanking a mutation region.The two allele-specific (inside) primers are designed in an oppositedirection to the common primers, both of wild-type and mutant ampliconsmay be simultaneously amplified with the common primers. As a result,two allele-specific amplicons may have different lengths since mutationsare asymmetrically located based on the common (outside) primers, andeasily separated by standard gel electrophoresis. The control ampliconsprovide an internal control for false negative results as well asamplification failure, and at least one of the two allele-specificamplicons is always present in the tetra-primer amplification-refractorymutation system PCR.

The “isothermal amplification” means that the amplification of a nucleicacid is not dependent on a thermocycler and is performed at a lowertemperature without the need for temperature change duringamplification. The temperature used in isothermal amplification mayrange from room temperature (22 to 24° C.) to approximately 65° C., orapproximately 60 to 65° C., 45 to 50° C., 37 to 42° C. or roomtemperature (22 to 24° C.). A product obtained by the isothermalamplification may be detected by gel electrophoresis, ELISA,enzyme-linked oligosorbent assay (ELOSA), real-time PCR, enhancedchemiluminescence (ECL), a chip-based capillary electrophoresis device,such as a bioanalyzer, for analyzing RNA, DNA and protein or turbidity.

In an exemplary embodiment of the present invention, E507K/R536K,E507K/R660V, or E507K/R536K/R660V Taq polymerase was used to confirmwhether an ability of extending a mismatched primer with respect to atemplate including a SNP (rs1408799, rs1015362 and/or rs4911414) wasreduced.

As a result, as shown in FIGS. 6 a-6 d , 7 and 8, compared to E507K Taqpolymerase, in the case of the E507K/R536K, E507K/R660V orE507K/R536K/R660V Taq polymerase, it can be confirmed that amplificationwith a mismatched primer is delayed, and such an effect was most clearlyshown in the case of the E507K/R536K/R660V Taq polymerase.

To this end, it was confirmed that the three types of DNA polymeraseshave higher mismatch extension selectivity than the conventional Taqpolymerase (E507K). Therefore, it is expected that the DNA polymerase ofthe present invention can be effectively used in medical diagnosis of adisease and recombinant DNA studies.

In another exemplary embodiment of the present invention, theE507K/R536K/R/R660V Taq polymerase was used to confirm whether anability of extending mismatched primers with respect to a template withQ61H, G13D or G12S SNP at the KRAS gene, and a template with L858R SNPat the EGFR gene was reduced.

As a result, as shown in FIGS. 10 a to 10 d , 11, 12 and 13, it wasconfirmed that Taq DNA polymerase having E507K/R536K/R587I/R660Vvariations, compared with Taq polymerase having E507K/R536K/R660Vvariations, has superior mismatch extension selectivity. Therefore, itis expected that the Taq DNA polymerase having E507K/R536K/R587I/R660Vvariations according to the present invention can also be effectivelyused in medical diagnosis of a disease and recombinant DNA studies.

The present invention also relates to a composition for detecting a genevariation or SNP, which includes the DNA polymerase according to thepresent invention, and a PCR kit including the same.

According to an exemplary embodiment of the present invention, the PCRkit may be applied to general PCR (first generation PCR), real-time PCR(second generation PCR), digital PCR (third generation PCR) orMassARRAY.

In the PCR kit of the present invention, the digital PCR may becompetitive allele-specific TaqMan PCR (CAST PCR) or droplet digital PCR(ddPCR), and more specifically, allele-specific cast PCR orallele-specific droplet digital PCR, but the present invention is notlimited thereto.

The “CAST PCR” is a method of detecting and quantifying rare mutationsfrom a large amount of sample containing normal wild-type gDNA, and toinhibit non-specific amplification from a wild-type allele, higherspecificity may be generated by the combination of allele-specificTaqMan® qPCR with an allele-specific MGB inhibitor, compared totraditional allele-specific PCR.

The “droplet digital PCR” is a system for counting target DNA after a 20μl PCR product is fractionated into 20,000 droplets and then amplified,and may be used to count positive droplets (1) and negative droplets (0)considered as digital signals according to the amplification of targetDNA in droplets, calculate the number of copies of target DNA by thePoisson distribution, and finally determine result values with thenumber of copies per μl sample, and used to detect rare mutations,amplify a very small amount of gene and simultaneously confirm amutation type.

The “MassARRAY” is a multiplexing analysis method that can be applied tovarious genome studies such as genotyping, using a MALDI-TOF massspectrometer, and may be used to rapidly analyze various samples andtargets at low cost or to perform customized analysis only for aspecific target.

The PCR kit of the present invention may include any reagent or otherelements, which are recognized for use in primer extension bytechnicians of ordinary skill in the art.

According to an exemplary embodiment of the present invention, the PCRkit may further include one or more matched primers, one or moremismatched primers or both of one or more matched primers and one ormore mismatched primers, wherein the one or more matched primers and oneor more mismatched primers are hybridized with a target sequence, andthe mismatched primer may include a non-canonical nucleotide at aposition of 7 bases from the 3′ end of the primer with respect to thehybridized target sequence.

The PCR kit of the present invention may further include a nucleosidetriphosphate.

The PCR kit of the present invention may further include a) one or morebuffers; b) a quantification reagent binding to double-stranded DNA; c)a polymerase blocking antibody, d) one or more control values or controlsequences; and e) one or more templates.

The present invention relates to a PCR buffer composition for increasingthe activity of a DNA polymerase with increased gene variationspecificity, which includes 25 to 100 mM KCl; and 1 to 15 mM (NH₄)₂SO₄,and has the final pH of 8.0 to 9.0.

Polymerases used in PCR should be optimal reaction buffers mixed withvarious materials to perform proper functions. The reaction buffersgenerally contain an element for pH stabilization, a metal ion as acofactor, and a stabilization element for preventing the denaturation ofa polymerase.

The KCl is an element required for enzyme stabilization, and helpspairing of a primer to target DNA. In the present invention, an optimalconcentration was determined by adjusting a KCl concentration in thereaction buffer to confirm a cation concentration in a state in whichthe amplification by mismatching is delayed as much as possible, and theamplification efficiency by matching is not reduced.

As a result of confirming an amplification delay effect by mismatchingaccording to the change in KCl concentration in the reaction bufferusing each of E507K, E507K/R536K, E507K/R660V and E507K/R536K/R660V Taqpolymerases, as shown in FIGS. 14 a to 14 d , the E507K/R536K/R660V Taqpolymerase showed an excellent amplification delay effect caused bymismatching without KCl in the reaction buffer, the E507K/R536K andE507K/R660V Taq polymerases showed an excellent amplification delayeffect caused by mismatching at 50 mM, and the control E507KTaqpolymerase showed an excellent amplification delay effect caused bymismatching at 100 mM. Consequently, it was confirmed that the KClconcentration threshold is the lowest for the E507K/R536K/R660V Taqpolymerase, and lower for the E507K/R536K and E507K/R660V Taqpolymerases, compared to that of E507K Taq polymerase.

In addition, to determine the optimal KCl concentration, theE507K/R536K/R660V Taq polymerase was used, and amplification wasperformed by variously changing a KCl concentration while a (NH₄)₂SO₄concentration was constantly fixed in the reaction buffer. As a resultof electrophoresis performed on an amplicon, as shown in FIG. 15 , theoptimal KCl concentration was confirmed to be 75 mM.

Therefore, the KCl concentration of the PCR buffer composition of thepresent invention may be 25 to 100 mM, preferably, 60 to 90 mM, morepreferably, 70 to 80 mM, and most preferably, 75 mM.

When the KCl concentration is less than 25 mM, it has no influence ongeneral target amplification, but a difference between amplification bya matched primer and amplification by a mismatched primer may bereduced, and when the KCl concentration is more than 100 mM, theefficiency of general target amplification may be lowered.

In the PCR buffer composition, the (NH₄)₂SO₄ is a cofactor required forenzyme activity, and used to increase polymerase activity along withTris. In an exemplary embodiment of the present invention, based on thedetermined results, the KCl concentration in the reaction buffer wasconstantly fixed at 75 mM, and the (NH₄)₂SO₄ concentration varied from2.5 mM to 25 mM, so that the optimal (NH₄)₂SO₄ concentration wasconfirmed.

As a result, as shown in FIG. 16 , at the (NH₄)₂SO₄ concentrationsranging from 2.5 to 15 mM, an amplicon was identified, confirming thatthe optimal (NH₄)₂SO₄ concentration was 5 mM.

In addition, by performing AS-qPCR at (NH₄)₂SO₄ concentrations ofapproximately 5 mM (2.5 mM, 5 mM and 10 mM), as shown in FIG. 17 , itwas confirmed that the Ct difference was the highest at 10 mM, but Ctwas a little delayed and a peak was tilted in the amplification causedby matching, and the optimal (NH₄)₂SO₄ concentration was determined tobe 5 mM.

Therefore, the (NH₄)₂SO₄ concentration in the PCR buffer composition maybe 1 to 15 mM, preferably, 2.5 to 8 mM, more preferably, 4 to 6 mM, andmost preferably, 5 mM.

When the (NH₄)₂SO₄ concentration is less than 1 mM, there was noinfluence on general target amplification, but the difference betweenthe amplification by a matched primer and the amplification by amismatched primer may be reduced, and when the (NH₄)₂SO₄ concentrationis more than 15 mM, the general target amplification efficiency may belowered.

Therefore, the optimized PCR buffer composition of the present inventionmay contain 70 to 80 mM KCl and 4 to 6 mM (NH₄)₂SO₄, and the final pH is8.0 to 9.0.

The PCR buffer composition of the present invention may further include5 to 80 mM tetra methyl ammonium chloride (TMAC).

TMAC is generally used to reduce amplification caused by mismatching orimprove the stringency of a hybridization reaction. In an exemplaryembodiment of the present invention, based on the obtained results, theoptimal TMAC concentration was determined by fixing the KClconcentration at 75 mM, and the (NH₄)₂SO₄ concentration at 5 mM in thereaction buffer, and varying a TMAC concentration from 0 to 80 mM.

As a result, as shown in FIGS. 18 a and 18 b , it was confirmed that theoptimal TAMC concentration was 70 mM for the E507K/R536K Taq polymerase,and 25 mM for the E507K/R536K/R660V Taq polymerase. In addition, as aresult of amplification performed by constantly fixing a TMACconcentration at 25 mM and a (NH₄)₂SO₄ concentration at 2.5 mM, andvarying a KCl concentration to 20, 40, 60 and 80 mM, as shown in FIGS.19 a and 19 b , the optimal KCl concentration for SNP rs1015362 andrs4911414 was determined to be 60 mM.

When the TMAC concentration is more than 80 mM, amplification efficiencyis reduced, and thus it is preferable that the TMAC concentration is inthe above-mentioned range.

Therefore, when the PCR buffer composition of the present inventioncontains 5 to 80 mM TMAC, the KCl concentration may be 40 to 90 mM, andpreferably, 50 to 80 mM, and the (NH₄)₂SO₄ concentration may be 1 to 7mM, and preferably 1.5 to 6 mM.

When TMAC is contained, the optimized PCR buffer composition of thepresent invention may contain 15 to 70 mM TMAC, 50 to 80 mM KCl, and 1.5to 6 mM (NH₄)₂SO₄, and the final pH may be 8.0 to 9.0.

The PCR buffer composition of the present invention may further containTris·Cl and MgCl₂, and additionally contain Tween 20 and bovine serumalbumin (BSA).

The present invention also provides a PCR kit for detecting a genevariation or SNP, which includes the above-described PCR buffercomposition.

The PCR kit of the present invention may further include a DNApolymerase comprising a Taq polymerase amino acid sequence of SEQ ID NO:1, where the DNA polymerase has substitution(s) of the following aminoacids:

-   -   (a) a substitution at amino acid residue 507 in the amino acid        sequence of SEQ ID NO: 1; and    -   (b) (i) a substitution at amino acid residue 536 in the amino        acid sequence of SEQ ID NO: 1,    -   (ii) a substitution at amino acid residue 660 in the amino acid        sequence of SEQ ID NO: 1,    -   (iii) substitutions at amino acid residues 536 and 660 in the        amino acid sequence of SEQ ID NO: 1, or    -   (iv) substitutions at amino acid residues 536, 587 and 660 in        the amino acid sequence of SEQ ID NO: 1.

According to an exemplary embodiment of the present invention, thesubstitution at the amino acid residue 507 may be a substitution ofglutamic acid (E) with lysine (K), the substitution at the amino acidresidue 536 may be a substitution of arginine (R) with lysine (K), thesubstitution at the amino acid residue 587 may be a substitution ofarginine (R) with isoleucine (I), and the substitution at the amino acidresidue 660 may be a substitution of arginine (R) with valine (V).

An additional description of the DNA polymerase included in the PCR kitof the present invention is the same as described above, and therefore,the overlapping description will be omitted.

Other components of the PCR kit for detecting a gene variation or SNP,which includes the PCR buffer composition of the present invention arethe same as described above, and the overlapping description will beomitted.

The present invention also relates to a method of in vitro detecting oneor more gene variations or SNPs using the kit for detecting a genevariation or SNP, which includes the PCR buffer composition forincreasing activity of the DNA polymerase with increased gene variationspecificity.

The method is the same as the method of in vitro detecting one or moregene variations or SNPs in one or more templates using the DNApolymerase with increased gene variation specificity of the presentinvention, except the components of the PCR buffer composition, andtherefore, the overlapping description will be omitted.

Hereinafter, the present invention will be described in further detailwith reference to examples. The examples are merely provided to morefully describe the present invention, and it will be obvious to those ofordinary skill in the art that the scope of the present invention is notlimited to the following examples.

EXAMPLES Example 11

Mutagenesis of Taq Polymerase

1-1. Fragment PCR

In this example, Taq DNA polymerase in which arginine was substitutedwith lysine at amino acid residue 536 in the amino acid sequence of SEQID NO: 1 (hereinafter, referred to as “R536K”), Taq DNA polymerase inwhich arginine was substituted with valine at amino acid residue 660 inthe amino acid sequence of SEQ ID NO: 1 (hereinafter, referred to as“R660V”) and Taq DNA polymerase in which arginine was substituted withlysine at amino acid residue 536 and arginine was substituted withvaline at amino acid residue 660 in the amino acid sequence of SEQ IDNO: 1 (hereinafter, referred to as “R536K/R660V”) were prepared asfollows.

First, using mutation-specific primers described in Table 1, as shown in(a) in FIG. 1 , Taq DNA polymerase fragments (F1 to F5) were amplifiedby PCR. Reaction conditions are as in Table 2.

TABLE 1  Primer Sequence (5′→3′) Eco-FGG GGTACC TCA TCA CCC CGG (SEQ ID NO: 17) R536K-RCTT GGT GAG CTC CTT GTA CTG CAG GAT  (SEQ ID NO: 18) R536K-FATC CTG CAG TAC AAG GAG CTC ACC AAG  (SEQ ID NO: 19) R660V-RGAT GGT CTT GGC CGC CAC GCG CAT CAG GGG  (SEQ ID NO: 20) R660V-FCCC CTG ATG CGC GTG GCG GCC AAG ACC ATC  (SEQ ID NO: 21) Xba-RGC TCTAGA CTA TCA CTC CTT GGC GGA GAG CCA (SEQ ID NO: 22)

TABLE 2 10 × pfu buffer (SolGent) 2.5 μl dNTP (10 mM each)  1 μl Fprimer (10 pmol/μl)  1 μl R primer (10 pmol/μl)  1 μl Distilled water 18μl pUC19-Tag (10 ng/μl)  1 μl Pfu polymerase 0.5 μl 30 cycles (Ta = 60°C.) 25 μl

PCR products were subjected to electrophoresis, thereby detecting a bandfor each fragment as shown in (b) in FIG. 1 , indicating that a desiredfragment was amplified.

1-2. Overlap PCR

Each amplified fragment obtained in 1-1 was used as a template, andfull-length amplification thereof was performed using primers at bothends (Eco-F and Xba-R primers). Reaction conditions are as in Tables 3and 4.

TABLE 3 R660V or R536K 10 × pfu buffer (SolGent)  5 μl 5 × enhancer(SolGent) 10 μl dNTP (10 mM each)  1 μl Eco-F primer (10 pmol/μl)  2 μlXba-R primer (10 pmol/μl)  2 μl Distilled water 27 μl Fragment 1 (orfragment 3)  1 μl Fragment 2 (or fragment 4)  1 μl Pfu polymerase  1 μl40 cycles (Ta = 62° C.) 50 μl

TABLE 4 R536K/R660V 10 × pfu buffer (SolGent)  5 μl 5 × enhancer(SolGent) 10 μl dNTP (10 mM each)  1 μl Eco-F primer (10 pmol/μl)  2 μlXba-R primer (10 pmol/μl)  2 μl Distilled water 26 μl Fragment 2  1 μlFragment 3  1 μl Fragment 5  1 μl Pfu polymerase  1 μl 40 cycles (Ta =62° C.) 50 μl

Consequently, as shown in (c) in FIG. 1 , it was confirmed that the Taqpolymerases of “R536K,” “R660V” and “R536K/R660V” were amplified.

1-3. Ligation

pUC19 was digested with restriction enzymes EcoRI/XbaI at 37° C. for 4hours under conditions shown in Table 5 below, DNA was purified, and thepurified DNA was treated with SAP at 37° C. for 1 hour under conditionsshown in Table 6, thereby preparing a vector.

TABLE 5 10 × CutSmart buffer (NEB)  2.5 μl pUC19 (500 ng/μl) 21.5 μlEcoRI-HF (NEB)  0.5 μl Xba I (NEB)  0.5 μl   25 μl

TABLE 6 10 × SAP buffer (Roche)  2 μl Purified DNA 17 μl SAP (Roche)  1μl 20 μl

After the overlap PCR product was obtained in Example 1-2 and digestedwith restriction enzymes EcoRI/XbaI at 37° C. for 3 hours underconditions shown in Table 7, an insert was gel-extracted with theprepared vector (FIG. 2 ).

TABLE 7 10 × CutSmart buffer (NEB)   2 μl Purified PCR product  17 μlEcoRI-HF (NEB) 0.5 μl XbaI (NEB) 0.5 μl  20 μl

After ligation was performed at room temperature for 2 hours underconditions shown in Table 8, E. coli DH5α was transformed with theresulting vectors and then screened in a medium containing ampicillin.Plasmids prepared from the collected colonies were sequenced, therebyobtaining Taq DNA polymerase mutants (“R536K,” “R660V” and“R536K/R660V”) into which desired variation(s) is/are introduced.

TABLE 8 Vector only Vector + Insert 10 × ligase buffer (SolGent) 1 μl 1μl Vector 1 μl 1 μl Insert — 3 μl Distilled water 7 μl 4 μl T4 DNAligase (SolGent) 1 μl 1 μl 10 μl  10 μl 

Example 21

Introduction of E507K Variation

2-1. Fragment PCR

The Taq polymerase activity of the “R536K,” “R660V” and “R536K/R660V”prepared in Example 1 was tested, thereby confirming that the activitywas decreased (data not shown), the E507K variation (substitution ofglutamic acid with lysine at amino acid residue 507 in the amino acidsequence of SEQ ID NO: 1) was additionally introduced into each ofR536K, R660V and R536K/R660V, and the E507K variation was introducedinto wild-type Taq DNA polymerase (WT) as a control. A method ofpreparing the E507K variation-introduced Taq DNA polymerase is the sameas described in Example 1.

Taq DNA polymerase fragments (F6 to F7) shown in Table 3 were amplifiedby PCR using mutation-specific primers shown in Table 9. Reactionconditions are shown in Table 10.

TABLE 9  Primer Sequence (5′→3′) Eco-FGG GGTACC TCA TCA CCC CGG (SEQ ID NO: 17) E507K-RCTT GCC GGT CTT TTT CGT CTT GCC GAT  (SEQ ID NO: 23) E507K-FATC GGC AAG ACG AAA AAG ACC GGC AAG  (SEQ ID NO: 24) Xba-RGC TCTAGA CTA TCA CTC CTT GGC GGA GAG CCA (SEQ ID NO: 22)

TABLE 10 10 × pfu buffer (SolGent) 2.5 μl dNTP (10 mM each)   1 μl Fprimer (10 pmol/μl)   1 μl R primer (10 pmol/μl)   1 μl Distilled water 18 μl Template plasmid (10 ng/μl)   1 μl Pfu polymerase 0.5 μl 30cycles (Ta = 60° C.)  25 μl

*Template plasmids: pUC19-Taq (WT), pUC19-Taq (R536K), pUC19-Taq(R660V), and pUC19-Taq (R536K/R660V)

2-2. Overlap PCR

Full-length amplification was performed on each of the amplifiedfragments obtained in 2-1 as a template using primers (Eco-F and Xba-Rprimers) at both ends. Reaction conditions are shown in Table 11.

TABLE 11 10X pfu buffer (SolGent)  5 μl 5X enhancer (SolGent) 10 μl dNTP(10 mM each)  1 μl Eco-F primer (10 pmol/μl)  2 μl Xba-R primer (10pmol/μl)  2 μl Distilled water 27 μl Fragment 6  1 μl Fragment 7  1 μlPfu polymerase  1 μl 40 cycles (Ta = 62° C.) 50 μl

2-3. Ligation

pUC19 was digested with restriction enzymes EcoRI/XbaI at 37° C. for 4hours under conditions shown in Table 5 above, DNA was purified, thepurified DNA was treated with SAP at 37° C. for 1 hour under conditionsshown in Table 6, thereby preparing a vector.

After the overlap PCR product was obtained in Example 2-2 and digestedwith restriction enzymes EcoRI/XbaI at 37° C. for 3 hours underconditions shown in Table 7, an insert was gel-extracted with theprepared vector (FIG. 4 ).

After ligation was performed at room temperature for 2 hours underconditions shown in Table 8, E. coli DH5α or DH10β was transformed withthe resulting vectors and then screened in a medium containingampicillin. Plasmids prepared from the collected colonies weresequenced, thereby obtaining E507K variation-introduced Taq DNApolymerase mutants (“E507K/R536K,” “E507K/R660V” and“E507K/R536K/R660V”).

Example 3

Performance of qPCR Using DNA Polymerase of the Present Invention

The Taq polymerase having each of the “E507K/R536K,” “E507K/R660V” and“E507K/R536K/R660V” variations obtained in Example 2 was used to confirmwhether an ability of extending a mismatched primer with respect to atemplate including a SNP was reduced. As a control, the “E507K” Taqpolymerase having the E507K variation was used.

The templates including SNPs used herein are rs1408799, rs1015362 andrs4911414, and genotypes of the templates and sequence data of specificprimers (IDT, USA) thereof are shown in Tables 12 and 13 below.

TABLE 12 Genotype of template rs1408799 TT rs1015362 CC rs4911414 GG

TABLE 13  Primer Name Sequence (5′→3′) rs1408799 Forward  CCAGTGTTAGGTTATTTCTAACTTG  (SEQ ID NO: 25) Reverse_TGCTCGGAGCACATGGTCAA  (SEQ ID NO: 26) Reverse_C GCTCGGAGCACATGGTCAG (SEQ ID NO: 27) rs1015362 Forward   TGAAGAGCAGGAAAGTTCTTCA (SEQ ID NO: 28) Reverse_C ACTGTGTGTCTGAAACAGTG  (SEQ ID NO: 29)Reverse_T ACTGTGTGTCTGAAACAGTA  (SEQ ID NO: 30) rs4911414 Forward_GGTAAGTCTTTGCTGAGAAATTCATTG  (SEQ ID NO: 31) Forward_TGTAAGTCTTTGCTGAGAAATTCATTT  (SEQ ID NO: 32) Reverse  AGTATCCAGGGTTAATGTGAAAG  (SEQ ID NO: 33)

Conditions for qPCR (Applied Biosystems 7500 Fast) are as shown in Table14 below.

TABLE 14 95° C. 5 min 95° C. 20 sec 50 cycles 60° C. 30 sec 72° C. 30sec 72° C. 3 min

Probes were dual-labeled as shown in Table 15 below.

TABLE 15  Probe 5′ 3′ Name Sequence (5′→3′) fluorophore quencher1408799- AGATATTTGTAAGGTATTCTG FAM Black Hole FAM GCCT (SEQ ID NO: 34)Quencher 1 1015362- TGCTGAACAAATAGTCCCGAC HEX Black Hole HEXCAG (SEQ ID NO: 35) Quencher 1 4911141- TTTCTCTAGTTGCCTTTAAGA Texas RedBlack Hole Texas  TTT (SEQ ID NO: 36) Quencher 2 Red

Oral epithelial cells were collected using a kit for collecting oralepithelial cells purchased from Noble Bio, lysed in 500 μl of a lysissolution, and then centrifuged at 12,000×g for 3 minutes. Thesupernatant was transferred to a fresh tube, and 1 μl per experiment wasused (FIG. 5 ). Reaction conditions are shown in Table 16, and thecomposition of the reaction buffer is shown in Table 17.

TABLE 16 5X reaction buffer 4 μl 5M betaine 2 μl dNTP (10 mM each) 0.5μl   Forward primer (2 μM) 1 μl Reverse primer (2 μM) 1 μl Nuclease-freedistilled water 8 μl Acquired template 1 μl Taq polymerase (2 U/μl) 0.5μl   Dual-labeled probe (4 μM) 2 μl 20 μl

TABLE 17 Reaction buffer (1X) 50 mM Tris-Cl (pH 8.8) 2.5 mM MgCl₂ 50 mMKCl 5 mM (NH₄)₂SO₄ 0.1% Tween 20 0.01% BSA

The other components of the reaction solution except a specific primerwere prepared as shown in Table 13 in two tubes, and eachallele-specific primer was added thereto, thereby performing qPCR. Here,a difference in cycle (Ct) value at which combined fluorescent signalsdetected from the tubes reach the threshold fluorescence valuecalculated with AB 7500 software (v2.0.6) was analyzed. It is consideredthat, as the Ct value in the amplification by a mismatched primer isdelayed, high gene variation specificity or allele specificity isexhibited. As a result of AS-qPCR for rs1408799, rs1015362 andrs4911414, as shown in FIGS. 6 a-6 d , 7 and 8, compared to the controlE507K, when the Tap polymerase having E507K/R536K, E507K/R660V orE507K/R536K/R660V variations was used, it was confirmed that theamplification by a mismatched primer was delayed, and such an effect wasmost significantly exhibited in the E507K/R536K/R660V mutant.

It was confirmed that the Tap DNA polymerase having the E507K/R536K,E507K/R660V or E507K/R536K/R660V variations according to the presentinvention, compared to that with E507K variation, has excellent mismatchextension selectivity. Therefore, it is expected that the three types ofTaq DNA polymerases can be useful for medical diagnosis of a disease andrecombinant DNA studies.

Example 4

Introduction of R587I Variation

4-1. Fragment PCR

To additionally introduce a R587I variation (substitution of argininewith isoleucine at amino acid residue 587 in the amino acid sequence ofSEQ ID NO: 1) into the “E507K/R536K/R660V” variation-introduced Taqclone prepared in Example 2, as shown in (a) in FIG. 9 , two fragmentswere amplified by PCR using primers shown in Table 18 below. Reactionconditions are shown in Table 19.

TABLE 18  Primer Sequence (5′-3′) Kpn-FTCC ACC CCG AGG GGT ACC TCA TCA CCC CGG CCT GGC (SEQ ID NO: 39) R5871-RCCC AAG CGG GGT GAT GAC GGG GAT GTT  (SEQ ID NO: 40) R5871-FAAC ATC CCC GTC ATC ACC CCG CTT GGG  (SEQ ID NO: 41) Xba-RCTG CAG GTC GAC TCT AGA CTA TCA CTC CTT GGC GGA G (SEQ ID NO: 42)

TABLE 19 10X pfu buffer (SolGent) 5 μl dNTP (10 mM each) 2 μl F primer(10 pmol/μl) 2 μl R primer (10 pmol/μl) 2 μl Distilled water 36 μl  Taqplasmid (E507K, R536K, R660V) 2 μl (10 ng/μl) Pfu polymerase 1 μl 35cycles (Ta = 60° C.) 50 μl 

The PCR product was confirmed by electrophoresis, and thus, as shown in(b) FIG. 9 , a band for each fragment was confirmed, indicating that adesired fragment was amplified.

4-2. In-Fusion Cloning

A Taq plasmid vector (E507K/R536K/R660V) was digested with restrictionenzymes KpnI/XbaI at 37° C. for 4 hours under conditions shown in Table20 and then purified (elution: 25 μl), thereby preparing an open linearvector. Afterward, an in-fusion cloning reaction was performed underconditions shown in Table 21 at 37° C. for 15 minutes to transform E.coli DH5α or DH10β, and then the transformed cell was screened in anampicillin-containing medium. Plasmids prepared from the collectedcolonies were sequenced, thereby obtaining a R587I variation-introducedTaq DNA polymerase mutant (“E507K/R536K/R587I/R660V”).

TABLE 20 10X CutSmart Vector (NEB) 2.5 μl Taq plasmid(E507K/R536K/R660V) 21.5 μl  (200 ng/μl) Kpn I-HF (NEB) 0.5 μl Xba I(NEB) 0.5 μl 25 μl

TABLE 21 5X EZ-fusio mix (Enzynomics) 2 μl Vector cleaved with Kpn I,Xba I 1 μl (50 ng/μl) F1 fragment (83 ng/μl) 1 μl F2 fragment (50 ng/μl)1 μl Distilled water 5 μl 10 μl

[Example 5] Performance of qPCR Using “E507K/R536K/R587I/R660V” TaqPolymerase

5-1. Discrimination of Q61H Variations in KRAS Gene

The Taq polymerase having the “E507K/R536K/R587I/R660V” variationsobtained in Example 4 was used to confirm whether an ability ofextending mismatched primers with respect to templates with Q61H SNPs inthe KRAS gene was reduced. As a control, the Taq polymerase having“E507K/R536K/R660V” variations was used.

The template including a SNP was gDNA (104 copies, 33 ng/rxn) obtainedfrom a HepG2 liver cancer cell line, and obtained by a typical DNAextraction method. It was confirmed that an entire detected target sitecorresponds to the NCBI reference sequence (NG_007524.1), and used as awild-type (WT).

The sequence data of specific primers for the template is shown in Table22 below.

TABLE 22  Primer Tm Name Sequence (5′-3′) (° C.) KRAS Forward_QGAT ATT CTC GAC ACA GCA 64.2 Q61H (24 mer) GGT CAA (SEQ ID NO: 43)Forward_H GAT ATT CTC GAC ACA GCA 64.4 (24 mer) GGT CAC (SEQ ID NO: 44)Reverse ACA AAG AAA GCC CTC CCC 64.2 AG (SEQ ID NO: 45)

Conditions for qPCR (Applied Biosystems 7500 Fast) are the same as shownin Table 14 in Example 3. Probes are labeled as shown in Table 23 below.

TABLE 23  Probe Tm Name Sequence (5′-3′) (° C.) Q61H FAMTGC AAT GAG GGA CCA GTA 67.6 CAT GAG G (SEQ ID NO: 46)

Reaction conditions are the same as shown in Table 16 in Example 3, andthe composition of the reaction buffer is the same as in Table 24 below.

TABLE 24 Reaction buffer (1X) 50 mM Tris-Cl (pH 8.8) 2.5 mM MgCl₂ 60 mMKCl 2.5 mM (NH₄)₂SO₄ 25 mM TMAC 0.1% Tween 20 0.01% BSA

The other components of the reaction solution except a specific primerwere prepared as shown in Table 22 in two tubes, and eachallele-specific primer was added thereto, thereby performing qPCR. Here,a difference in cycle (Ct) value at which combined fluorescent signalsdetected from the tubes reach the threshold fluorescence valuecalculated with AB 7500 software (v2.0.6) was analyzed. It is consideredthat, as the Ct value in the amplification by a mismatched primer isdelayed, high gene variation specificity or allele specificity isexhibited. As a result of AS-qPCR, as shown in FIGS. 10 a and 10 b ,compared to the control E507K/R536K/R660V, the Taq polymerase havingE507K/R536K/R587I/R660V variations was increased in ΔCt up to 5,indicating that the amplification by a mismatched primer was delayed.

The inventors further performed the above-described experiment onceagain using a primer shown in Table 25, which was manufactured byshortening the 24-mer primer of Table 22 to 18 mer. Except for using thecomposition of the reaction buffer in Table 26 below, all conditions arethe same as those in the experiment using the 24-mer primer.

TABLE 25  Primer  Tm Name Sequence (5′-3′) (° C.) KRAS  Forward_QCTC GAC ACA GCA GGT CAA 61.4 Q61H (18 mer) (SEQ ID NO: 47) Forward_HCTC GAC ACA GCA GGT CAC 61.8 (18 mer) (SEQ ID NO: 48) ReverseACA AAG AAA GCC CTC CCC 64.2 AG (SEQ ID NO: 49)

TABLE 26 Reaction buffer (1X) 50 mM Tri-Cl (pH 8.8) 2.5 mM MgCl₂ 15 mM(NH₄)₂SO₄ 0.1% Tween 20 0.01% BSA

Consequently, as shown in FIGS. 10 c and 10 d , compared to the controlE507K/R536K/R660V, the Taq polymerase having E507K/R536K/R587I/R660Vvariations can confirm that the amplification by a mismatched primer isdelayed. Particularly, the ΔCt of the R587I-introduced polymerase wasmore remarkably increased.

5-2. Discrimination of G13D Variations in KRAS Gene

The Taq polymerase having the “E507K/R536K/R587I/R660V” variationsobtained in Example 4 was used to confirm whether an ability ofextending mismatched primers with respect to templates with G13D SNPs inthe KRAS gene was reduced. As a control, Taq polymerase having“E507K/R536K/R660V” variations was used.

The template including an SNP was gDNA (104 copies, 33 ng/rxn) obtainedfrom a HepG2 liver cancer cell line, and obtained by a typical DNAextraction method. It was confirmed that an entire detected target sitecorresponds to the NCBI reference sequence (NG_007524.1), and used as awild-type (WT).

The sequence data of specific primers for the template is shown in Table27 below.

TABLE 27  Primer Tm Name Sequence (5′-3′) (° C.) KRAS ForwardATA AGG CCT GCT GAA AAT GAC  61 G13D (SEQ ID NO: 50) Reverse_GGGC ACT CTT GCC TAC GC  62.4 (17mer) (SEQ ID NO: 51) Rev erse_DGGC ACT CTT GCC TAC GT  61.2 (17 mer) (SEQ ID NO: 52)

Conditions for qPCR (Applied Biosystems 7500 Fast) are the same as shownin Table 14 in Example 3. Probes are labeled as shown in Table 28 below.

TABLE 28  Probe  Tm Name Sequence (5′-3′) (° C.) G1213_RAGC TCC AAC TAC CAC AAG TTT ATA  66.2 FAM TTC AGT (SEQ ID NO: 53)

Reaction conditions are the same as shown in Table 16 in Example 3, andthe composition of the reaction buffer is the same as in Table 24 inExample 5-1. The other components of the reaction solution except aspecific primer were prepared as shown in Table 27 in two tubes, andeach allele-specific primer was added thereto, thereby performing qPCR.Here, a difference in cycle (Ct) value at which combined fluorescentsignals detected from the tubes reach the threshold fluorescence valuecalculated with AB 7500 software (v2.0.6) was analyzed. It is consideredthat, as the Ct value in the amplification by a mismatched primer isdelayed, high gene variation specificity or allele specificity isexhibited.

As a result of AS-qPCR, as shown in FIG. 11 , compared to the controlE507K/R536K/R660V, the Taq polymerase having E507K/R536K/R587I/R660Vvariations confirmed that the amplification by a mismatched primer wasdelayed.

5-3. Discrimination of G12S Variations in KRAS Gene

The Taq polymerase having the “E507K/R536K/R587I/R660V” variationsobtained in Example 4 was used to confirm whether an ability ofextending mismatched primers with respect to templates having GI3S SNPsin the KRAS gene was reduced. As a control, the Taq polymerase having“E507K/R536K/R660V” variations was used.

The template having an SNP was gDNA (104 copies, 33 ng/rxn) obtainedfrom a HepG2 liver cancer cell line, and obtained by a typical DNAextraction method. It was confirmed that an entire detected target sitecorresponds to the NCBI reference sequence (NG_007524.1), and used as awild-type (WT).

The sequence data of specific primers for the template is shown in Table29 below.

TABLE 29  Primer Tm Name Sequence (5′-3′) (° C.) KRAS Forward_GTAA ACT TGT GGT AGT TGG  62.6 G125 (23 mer) AGC TG (SEQ ID NO: 54)Forward_S TAA ACT TGT GGT AGT TGG  61.6 (23 mer) AGC TA (SEQ ID NO: 55)Reverse CAT ATT CGT CCA CAA AAT  63 GAT TCT GAA T (SEQ ID  NO: 56)

Conditions for qPCR (Applied Biosystems 7500 Fast) are the same as shownin Table 14 in Example 3. Probes are labeled as shown in Table 30 below.

TABLE 30  Tm Probe Name Sequence (5′-3′) (° C.) G1213_F FAMAGC TGT ATC GTC AAG GCA  68.2 CTC TTG C (SEQ ID NO: 57)

Reaction conditions are the same as shown in Table 16 in Example 3, andthe composition of the reaction buffer is the same as in Table 24 inExample 5-1. The other components of the reaction solution except aspecific primer were prepared as shown in Table 29 in two tubes, andeach allele-specific primer was added thereto, thereby performing qPCR.Here, a difference in cycle (Ct) value at which combined fluorescentsignals detected from the tubes reach the threshold fluorescence valuecalculated with AB 7500 software (v2.0.6) was analyzed. It is consideredthat, as the Ct value in the amplification by a mismatched primer isdelayed, high gene variation specificity or allele specificity isexhibited.

As a result of AS-qPCR, as shown in FIG. 12 , compared to the controlE507K/R536K/R660V, the Taq polymerase having E507K/R536K/R587I/R660Vvariations confirmed that the amplification by a mismatched primer wasdelayed.

5-4. Discrimination of L858R Variations in EGFR Gene

The Taq polymerase having the “E507K/R536K/R587I/R660V” variationsobtained in Example 4 was used to confirm whether an ability ofextending mismatched primers with respect to templates with L858R SNPsin EGFR gene was reduced. As a control, Taq polymerase having“E507K/R536K/R660V” variations was used.

The template including a SNP was gDNA (104 copies, 33 ng/rxn) obtainedfrom a HepG2 liver cancer cell line, and obtained by a typical DNAextraction method. It was confirmed that an entire detected target sitecorresponds to the NCBI reference sequence (NG_007726.3), and used as awild-type (WT).

The sequence data of specific primers for the template is shown in Table31 below.

TABLE 31  Primer  Name Sequence (5′-3′) Tm (° C.) EGFR  ForwardACC TGG CAG CCA GGA  67.8 L858R ACG TA (SEQ ID NO: 58) Reverse_LGCA CCC AGC AGT TTG  68.2 GCC A (SEQ ID NO: 59) Reverse_RGCA CCC AGC AGT TTG  67.7 GCC C (SEQ ID NO: 60)

Conditions for qPCR (Applied Biosystems 7500 Fast) are the same as shownin Table 14 in Example 3. Probes are labeled as shown in Table 32 below.

TABLE 32  Probe Name Sequence (5′-3′) Tm (° C.) L858R FAM_RCAG CAT GTC AAG ATC ACA GAT 67.8 TTT GGG C (SEQ ID NO: 61)

Reaction conditions are the same as shown in Table 16 in Example 3, andthe composition of the reaction buffer is the same as in Table 24 inExample 5-1. The other components of the reaction solution except aspecific primer were prepared as shown in Table 31 in two tubes, andeach allele-specific primer was added thereto, thereby performing qPCR.Here, a difference in cycle (Ct) value at which combined fluorescentsignals detected from the tubes reach the threshold fluorescence valuecalculated with AB 7500 software (v2.0.6) was analyzed. It is consideredthat, as the Ct value in the amplification by a mismatched primer isdelayed, high gene variation specificity or allele specificity isexhibited.

As a result of AS-qPCR, as shown in FIG. 13 , compared to the controlE507K/R536K/R660V, the Taq polymerase having E507K/R536K/R587I/R660Vvariations confirmed that the amplification by a mismatched primer wasdelayed.

As described above, it was confirmed that some of the Taq DNApolymerases having E507K/R536K/R587I/R660V variations according to thepresent invention, compared to the Taq polymerase havingE507K/R536K/R660V variations, have excellent mismatch extensionselectivity. Therefore, the Taq DNA polymerases havingE507K/R536K/R587I/R660V variations according to the present inventionare also expected to be usefully applied to the medical diagnosis of adisease and recombinant DNA studies.

Example 61

Optimization of KCl Concentration in Reaction Buffer

In this example, to find a high cation concentration in a state in whichthe amplification by mismatching is delayed as much as possible, and theamplification efficiency by matching is not reduced, an optimal KClconcentration was confirmed by adjusting a KCl concentration in a PCRbuffer.

The Taq polymerases having “E507K/R536K,” “E507K/R660V” and“E507K/R536K/R660V” variations, respectively, obtained in Example 2 wereused to compare a KCl concentration threshold with the Taq polymerasehaving the E507K variation.

As a template having an SNP, rs1408799 was used, the genotype of thetemplate was TT, and as a primer, an rs1408799 primer shown in Table 2was used. qPCR (Applied Biosystems 7500 Fast) was performed under theconditions shown in Table 14, a dual-labeled probe is 1408799-FAM shownin Table 15, the reaction conditions are shown in Table 33, and thecomposition of the reaction buffer is shown in Table 34.

TABLE 33 5X Reaction buffer 4 μl dNTP (10 mM each) 0.5 μl   Forwardprimer (2 μM) 1 μl Reverse primer (2 μM) 1 μl Nuclease-free distilledwater 10 μl   Acquired template (TT) 1 μl Taq polymerase (2 U/μl) 0.5μl   Dual-labeled probe (4 μM) 2 μl 20 μl

TABLE 34 Reaction buffer (1X) 50 mM Tris-Cl (pH 8.8) 2.5 mM MgCl₂ x mMKCl 2.5 mM (NH₄)₂SO₄ 0.1% Tween 20 0.01% BSA

Consequently, as shown in FIGS. 14 a to 14 d , it was confirmed that theE507K/R536K/R660V Taq polymerase has the lowest KCl concentrationthreshold, and the E507K/R536K and E507K/R660V have lower KClconcentration thresholds than E507K. Based on the result, to determinethe optimal KCl concentration, an additional experiment was performedusing the E507K/R536K/R660V Taq polymerase. A primer was thers1408799-T-specific primer shown in Table 13, qPCR (Applied Biosystems7500 Fast) was performed for 35 cycles under the conditions shown inTable 14, and reaction conditions are shown in Table 35.

TABLE 35 5X Reaction buffer 4 μl dNTP (10 mM each) 0.5 μl   Forwardprimer (2 μM) 1 μl Reverse primer (2 μM) 1 μl Nuclease-free distilledwater 12 μl   Acquired template (TT) 1 μl E507K/R536K/R660V (2 U/μl) 0.5μl   20 μl

The composition of a reaction buffer for the control is shown in Table36, and the composition of a reaction buffer for the experimental groupis shown in Table 34. The (NH₄)₂SO₄ concentration was constantly fixedat 2.5 mM, and the KCl concentration varied.

TABLE 36 Control buffer (1X) 50 mM Tris-Cl (pH 8.8) 1M betaine 2.5 mMMgCl₂ 50 mM KCl 2.5 mM (NH₄)₂SO₄ 0.1% Tween 20 0.01% BSA

Amplification was performed under the above-mentioned conditions, andthe PCR product was identified by electrophoresis, thereby confirmingthat, as shown in FIG. 15 , an optimal KCl concentration in a state inwhich the amplification by mismatching is delayed as much as possible,and the amplification efficiency by matching is not reduced is 75 mM.

Example 7

Optimization of (NH₄)₂SO₄ Concentration in Reaction Buffer

In this example, based on the result of Example 4, the optimal (NH₄)₂SO₄concentration was confirmed by constantly fixing a KCl concentration ina reaction buffer at 75 mM and variously changing a (NH₄)₂SO₄concentration. As a primer, the rs1408799-T-specific primer shown inTable 13 was used. qPCR (Applied Biosystems 7500 Fast) was performed for35 cycles under the conditions shown in Table 14, reaction conditionsare shown in Table 35, and the composition of a reaction buffer for thecontrol is shown in Table 36.

Consequently, as shown in FIG. 16 , it was confirmed that an optimal(NH₄)₂SO₄ concentration is 5 mM.

Based on the result, an amplification delay effect caused by mismatchingwas further confirmed by constantly fixing the KCl concentration in thereaction buffer at 75 mM, and setting the (NH₄)₂SO₄ concentration toapproximately 5 mM (each of 2.5 mM, 5 mM and 10 mM).

As a primer, the rs1408799 primer shown in Table 13 was used, adual-labeled probe is 1408799-FAM shown in Table 15, and reactionconditions are shown in Table 37 below.

TABLE 37 5X Reaction buffer 4 μl dNTP (10 mM each) 0.5 μl   Forwardprimer (2 μM) 1 μl Reverse primer (2 μM) 1 μl Nuclease-free distilledwater 10 μl  Acquired template (TT) 1 μl E507K/R536K/R660V (2 U/μl) 0.5μl   Dual-labeled probe (4 μM) 2 μl 20 μl

Consequently, as shown in FIG. 17 , when the (NH₄)₂SO₄ concentration was10 mM, the Ct value difference was the largest, but Ct was a littledelayed and a peak was tilted in the amplification caused by matching,and the optimal (NH₄)₂SO₄ concentration was determined to be 5 mM. Bycombining the results of Examples 6 and 7, it was confirmed that theoptimal composition of a reaction buffer contains 50 mM Tris-Cl, 2.5 mMMgCl₂, 75 mM KCl, 5 mM (NH₄)₂SO₄, 0.1% Tween 20 and 0.01% BSA.

Example 81

Addition of TMAC to Reaction Buffer and Optimization of TMACConcentration

In this example, the optimal concentration was confirmed by adding TMACto a reaction buffer. Based on the results of Examples 6 and 7, theoptimal TMAC concentration was determined by constantly fixing a KClconcentration at 75 mM and a (NH₄)₂SO₄ concentration at 5 mM, andvariously changing a TMAC concentration.

A E507K/R536K or E507K/R536K/R660V Taq polymerase was used, and as atemplate having an SNP, rs1408799 was used. The genotype of the templatewas TT, and as a primer, the rs1408799 primer shown in Table 13 wasused. qPCR (Applied Biosystems 7500 Fast) was performed under theconditions shown in Table 14, a dual-labeled probe is 1408799-FAM shownin Table 15, and reaction conditions are shown in Table 37.

Consequently, as shown in FIGS. 18 a and 18 b , it was confirmed that,for the E507K/R536K Taq polymerase, the optimal TMAC concentration is 60mM, and for the E507K/R536K/R660V Taq polymerase, the optimal TMACconcentration is 25 mM. When the TMAC concentration is very high,amplification efficiency was reduced.

Example 91

Optimization of KCl, (NH₄)₂SO₄ and TMAC Concentrations in ReactionBuffer

In this Example, based on the result shown in Example 8, the optimalKCl, (NH₄)₂SO₄ and TMAC concentrations in a reaction buffer wereconfirmed using the E507K/R536K/R660V Taq polymerase.

Specifically, the TMAC concentration was constantly fixed at 25 mM, the(NH₄)₂SO₄ concentration was constantly fixed at 2.5 mM, and then the KClconcentration was changed to 20, 40, 60 or 80 mM. An experiment wasperformed on two SNPs of rs1015362 and rs4911414, and the genotype ofthe template is shown in Table 12, primers were the rs1015362 andrs4911414 primers shown in Table 13, qPCR (Applied Biosystems 7500 Fast)was performed under the conditions shown in Table 14, a dual-labeledprobe is 1408799-FAM shown in Table 15, and reaction conditions areshown in Table 37.

Consequently, as shown in FIGS. 19 a and 19 b the optimal KClconcentration for two SNPs was 60 mM, and it can be observed that whenthe KCl concentration was 80 mM, amplification efficiency was reduced.

From the above-described results, it was confirmed that the optimal KClconcentration in the reaction buffer was 60 mM, the optimal (NH₄)₂SO₄concentration was 2.5 mM, and the optimal TMAC concentration was 25 mM,and in further detail, for the E507K/R536K polymerase, 75 mM KCl, 5 mM(NH₄)₂SO₄ and 60 mM TMAC were most effectively used, and for theE507K/R536K/R660V polymerase, 60 mM KCl, 2.5 mM (NH₄)₂SO₄ and 25 mM TMACwere most effectively used.

INDUSTRIAL APPLICABILITY

Since the DNA polymerase with increased gene variation specificityaccording to the present invention has a higher mismatch-to-matchextension selectivity than conventional Taq polymerase, reliable genevariation-specific amplification is possible without any substratemodification. The present invention provides an optimal PCR buffercomposition that allows the proper function of a DNA polymerase withincreased gene variation specificity to be effectively exhibited, andreliable gene variation-specific amplification is possible byconsiderably increasing the activity of the DNA polymerase using the DNApolymerase with increased gene variation specificity. Moreover, a kitincluding a PCR buffer composition and/or the DNA polymerase withincreased gene variation specificity according to the present inventioncan effectively detect a gene variation or SNP, and thus can be usefullyapplied to the medical diagnosis of a disease and recombinant DNAstudies.

The invention claimed is:
 1. A Taq polymerase mutant wherein the Taqpolymerase mutant consists of the amino acid sequence of SEQ ID NO: 8that has 3 amino acid substitutions in SEQ ID NO: 1, wherein glutamicacid (E) at position 507 of SEQ ID NO:1 is substituted with lysine (K)arginine (R) at position 536 of SEQ ID NO:1 is substituted with lysine(K), and arginine (R) at position 660 of SEQ ID NO:1 is substituted withvaline (V), and wherein the mutant has Taq polymerase activity.
 2. TheTaq polymerase mutant of claim 1, wherein the Taq polymerase mutant isused for discriminating a matched primer from a mismatched primer bycomparing a Ct value of a quantitative polymerase chain reaction (qPCR)in the presence of a nucleic acid template and the matched primer with aCt value of a qPCR in the presence of the nucleic acid template and themismatched primer, wherein both the matched primer and the mismatchedprimer are capable of hybridizing with a target sequence of the nucleicacid template, and the mismatched primer has a non-complementarynucleotide at its 3′ end after it hybridizes to the target sequence ofthe nucleic acid template; wherein the matched primer consisting of anucleotide sequence of SEQ ID NO: 26, the mismatched primer consistingof a nucleotide sequence of SEQ ID NO: 27, and the nucleic acid templateis a nucleic acid comprising a single nucleotide polymorphisms (SNP)rs1408799; or wherein the matched primer consisting of a nucleotidesequence of SEQ ID NO: 29, the mismatched primer comprises a nucleotidesequence of SEQ ID NO: 30, and the nucleic acid template is a nucleotideacid comprising a SNP rs1015362; or wherein the matched primerconsisting of a nucleotide sequence of SEQ ID NO: 31, the mismatchedprimer consisting of a nucleotide sequence of SEQ ID NO: 32, and thenucleic acid template is a nucleotide acid comprising a SNP rs4911414.3. A method of in vitro detecting a single nucleotide polymorphisms(SNP) in a nucleic acid template, the method comprising: conducting aqPCR in the presence of the Taq polymerase mutant DNA polymerase ofclaim 1 and the nucleic acid template.
 4. A PCR kit comprising the Taqpolymerase mutant of claim 1, a matched primer and a mismatched primer,wherein the matched primer and the mismatched primer are hybridized witha target sequence.
 5. A PCR kit comprising the Taq polymerase mutant ofclaim 1 and a nucleoside triphosphate.
 6. A PCR kit comprising: (a) theTaq polymerase mutant DNA polymerase of claim 1; (b) one or morebuffers; (c) a quantification reagent binding to a double-stranded DNA;(d) a polymerase blocking antibody; (e) one or more control values orcontrol sequences; and (f) one or more nucleic acid templates.
 7. A Taqpolymerase mutant, wherein the Taq polymerase mutant consists of theamino acid sequence of SEQ ID NO: 37 that has 4 amino acid substitutionsin SEQ ID NO: 1, wherein glutamic acid (E) at position 507 of SEQ ID NO:1 is substituted with lysine (K), arginine (R) at position 536 of SEQ IDNO: 1 is substituted with lysine (K), arginine (R) at position 587 issubstituted with isoleucine (I), and arginine (R) at position 660 of SEQID NO: 1 is substituted with valine (V), and wherein the mutant has Taqpolymerase activity.
 8. The Taq polymerase mutant of claim 7, whereinthe Taq polymerase mutant is used for discriminating a matched primerfrom a mismatched primer by comparing a Ct value of a quantitativepolymerase chain reaction (qPCR) in the presence of a nucleic acidtemplate and the matched primer with a Ct value of a qPCR in thepresence of the nucleic acid template and the mismatched primer, whereinboth the matched primer and the mismatched primer are capable ofhybridizing with a target sequence of the nucleic acid template, and themismatched primer has a non-complementary nucleotide at its 3′ end afterit hybridizes to the target sequence of the nucleic acid template;wherein the matched primer consists of a nucleotide sequence of SEQ IDNO: 43 and the mismatched primer consists of a nucleotide sequence ofSEQ ID NO: 44 when the nucleic acid template is a KRAS gene comprisingQ61H variation, or the matched primer consists of a nucleotide sequenceof SEQ ID NO: 47 and the mismatched primer consists of a nucleotidesequence of SEQ ID NO: 48 when the nucleic acid template is a KRAS genecomprising Q61H variation; or wherein the matched primer consists of anucleotide sequence of SEQ ID NO: 51 and the mismatched primer consistsof a nucleotide sequence of SEQ ID NO: 52 when the nucleic acid templateis a KRAS gene comprising G13D variation; or wherein the matched primerconsists of a nucleotide sequence of SEQ ID NO: 54 and the mismatchedprimer consists of comprises a nucleotide sequence of SEQ ID NO: 55 whenthe nucleic acid template is a KRAS gene comprising G12S variation; orwherein the matched primer consists of a nucleotide sequence of SEQ IDNO: 59 and the mismatched primer consists of a nucleotide sequence ofSEQ ID NO: 60 when the nucleic acid template is a EGFR gene comprisingL585R variation.
 9. A method of in vitro detecting an SNP in a template,the method comprising: conducting a qPCR in the presence of the Taqpolymerase mutant DNA polymerase of claim 2 and the nucleic acidtemplate.
 10. A PCR kit comprising the Taq polymerase mutant of claim 7,a matched primer and a mismatched primer, wherein the matched primer andthe mismatched primer are hybridized with a target sequence.
 11. A PCRkit comprising the Taq polymerase mutant of claim 7 and a nucleosidetriphosphate.
 12. A PCR kit comprising: (a) the Taq polymerase mutantDNA polymerase of claim 7; (b) a nucleoside triphosphate; (c) one ormore buffers; (d) a quantification reagent binding to a double-strandedDNA; (e) a polymerase blocking antibody; (f) one or more control valuesor control sequences; and (g) one or more nucleic acid templates.
 13. Amethod of performing a competitive allele-specific TaqMan (CAST) PCR, adroplet digital PCR or a mass spectrometry using the PCR kit of claim 4,wherein the mismatched primer in the PCR kit is a mutant allele-specificprimer, and wherein the PCR kit further comprises a wild typeallele-specific blocker.
 14. A method of performing a CAST PCR, adroplet digital PCR or a mass spectrometry using the PCR kit of claim10, wherein the mismatched primer in the PCR kit is a mutantallele-specific primer, and wherein the PCR kit further comprises a wildtype allele-specific blocker.